PIK3CA Mutation Status and SASH1 Expression Predicts Synergy Between Lapatinib and an AKT Inhibitor in HER2 Positive Breast Cancer

ABSTRACT

Methods for identifying a cancer patient, such as a breast cancer patient, suitable for treatment with a 4-anilinoquinazoline kinase inhibitor, such as lapatinib, and an AKT inhibitor, comprising detecting modulated expression of HER2 (ERBB2) and SASH1 or protein encoded thereof and detecting PIK3CA mutation status. High levels of expression in HER2 and high levels of SASH1 and/or positive PIK3CA mutation status indicate a patient that is suitable for treatment with a 4-anilinoquinazoline kinase inhibitor, such as lapatinib and an AKT inhibitor.

STATEMENT OF GOVERNMENTAL SUPPORT

The invention described was made with government support under ContractNo. DE-AC02-05CH11231 awarded by the U.S. Department of Energy, underWork for Others Agreement No. LB06002417, and under Grant No. CA 126551SPORE grant and Grant No. P50 CA58207 awarded by the National Institutesof Health. The government has certain rights in this invention.

INCORPORATION OF SEQUENCE LISTING AND TABLES

The application includes and incorporates the attached sequence listingand Tables 1 and 2.

FIELD OF THE INVENTION

This invention relates generally to genetic markers involved in thediagnosis and prognosis of ERBB2/HER2-positive cancer (HER2 positive),predicting patient response to specific therapeutic compounds andproviding such therapy to patients predicted to benefit from suchtherapy.

BACKGROUND OF THE INVENTION

HER2 amplification occurs in approximately 20% of all breast cancerpatients. Therapeutic agents such as trastuzumab and lapatinib have beendeveloped that target this alteration. Unfortunately, many patients withHER2 amplification are non-responsive to these drugs, or developresistance to the drugs. Currently, patients who are non-responsive ordevelop resistance to Lapatinib are put onto experimental trials withother chemotherapeutic agents for treatment of advanced breast cancer.

Lapatinib is currently given to patients with metastatic HER2 positivebreast cancer in combination with traditional chemotherapeutic agentssuch as paclitaxel.

Recent evidence suggests that activation of the PI3K-AKT pathway mayoccur following inhibition of HER2. This suggests that targeting thispathway in combination with HER2 inhibition may be synergistic and thushave therapeutic benefit.

SUMMARY OF THE INVENTION

The invention provides for a method for identifying a HER2-positivecancer patient suitable for treatment with a 4-anilinoquinazoline kinaseinhibitor Lapatinib and an AKT inhibitor, comprising: (a) obtaining thesequence of the PIK3CA gene, mRNA, or protein from a sample from apatient; and (b) identifying any mutations in the PIK3CA gene or proteinin a sample from the patient as compared to the wild-type sequence foundin SEQ ID NOs:3, 4, or 11, wherein a mutation in the PIK3CA gene, mRNA,or protein indicates the patient is suitable for treatment with the4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.

The method, further comprising (c) measuring the genomic copy number orexpression level of a gene encoding HER2 in a sample from the patient,and (d) comparing the HER2 genomic copy number in the patient to normalcopy number or expression level of the gene encoding HER2, theexpression level of the gene encoding HER2 in a normal tissue sample ora reference expression level, or the average expression level of HER2 ina panel of normal cell lines or cancer cell lines, wherein an increasein the expression level of HER2 indicates the patient is suitable fortreatment with the 4-anilinoquinazoline kinase inhibitor and an AKTinhibitor. In another embodiment, the method, (c) measuring the HER2protein levels in a sample from the patient, and (d) comparing the HER2protein levels from the sample to normal HER2 protein levels in a normaltissue sample or a reference protein level, or the average protein levelof HER2 in a panel of normal cell lines or cancer cell lines, wherein anincrease in the protein levels of HER2 indicates the patient is suitablefor treatment with the 4-anilinoquinazoline kinase inhibitor and an AKTinhibitor. Patients identified by the present invention will respond tothe synergistic treatment of cancer with the 4-anilinoquinazoline kinaseinhibitor and an AKT inhibitor.

In some embodiments of the invention, a method for identifying aHER2-positive cancer patient suitable for treatment with a4-anilinoquinazoline kinase inhibitor and an AKT inhibitor, comprising:(a) measuring the expression level of the SASH1 gene in a sample fromthe patient; and (b) comparing the expression level of said gene fromthe patient with the expression level of the gene in a normal tissuesample or a reference expression level (such as the average expressionlevel of the gene in a cell line panel or a cancer cell or tumor panel,or the like), wherein an increase in the expression level of SASH1indicates the patient is suitable for treatment with the4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.

In some embodiments of the invention, the patient identified as suitablefor treatment with a combination of 4-anilinoquinazoline kinaseinhibitor and an AKT inhibitor. In other embodiments, the4-anilinoquinazoline kinase inhibitor and an AKT inhibitor areadministered concurrently or sequentially.

In another embodiment, a method for identifying a HER2-positive cancerpatient suitable for treatment with a 4-anilinoquinazoline kinaseinhibitor and an AKT inhibitor, comprising: (a) obtaining the sequenceof the PIK3CA gene from a sample from a patient; (b) identifying anymutations in the PIK3CA gene in a sample from the patient as compared tothe wild-type sequence found in SEQ ID NOs:3, 4 or 11, (c) measuring theexpression level of the SASH1 gene in the sample from the patient; and(d) comparing the expression level of said gene from the patient withthe expression level of the gene in a normal tissue sample or areference expression level (such as the average expression level of thegene in a cell line panel or a cancer cell or tumor panel, or the like),wherein both a mutation in the PIK3CA gene and an increase in theexpression level of SASH1 indicates the patient is suitable fortreatment with the 4-anilinoquinazoline kinase inhibitor and an AKTinhibitor.

The invention provides for a method of treating a cancer patientcomprising (a) identifying a cancer patient who is suitable fortreatment with a 4-anilinoquinazoline kinase inhibitor and an AKTinhibitor using a method of the present invention, and (b) administeringa therapeutically effective amount of the 4-anilinoquinazoline kinaseinhibitor and the AKT inhibitor to the patient. In some embodiments, acombination of the 4-anilinoquinazoline kinase inhibitor and an AKTinhibitor, and in other embodiments, the 4-anilinoquinazoline kinaseinhibitor and an AKT inhibitor are administered concurrently orsequentially.

In some embodiments, the cancer is breast cancer and the cancer patientis a breast cancer patient. In certain embodiments, the breast cancerpatient is an ERBB2-positive breast cancer patient.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

The foregoing aspects and others will be readily appreciated by theskilled artisan from the following description of illustrativeembodiments when read in conjunction with the accompanying drawings.

FIG. 1 shows representative examples of HER2 positive breast cancer celllines subtypes and their response to an AKT inhibitor (triangle),lapatinib (square), or a combination of the two (diamond). Y-axisrepresents % growth inhibition, X-axis represents dose. Each graph islabeled with the cell line name and the PIK3CA mutation found.

FIG. 2 shows a heatmap showing synergy across nine different doses of a1:1 combinations of Lapatinib+AKT inhibitor (light gray indicatessignificant synergy, black indicates additivity/no significantinteraction, and dark gray indicates antagonism). Note synergisticinteractions between the inhibitors in the cell lines with PI3K pathwaymutations, while those without PI3K mutations show additivity orantagonism.

FIG. 3 shows relative SASH1 expression levels in breast cancer celllines that show synergy with lapatinib+AKT ((HCC202; MDAMB361; HCC1954;SUM190PT; HCC1569)) compared to those that are notsynergistic/antagonistic (AU565; MDAMB453; SKBR3; SUM225CWN; UACC812)with the combination. BT474 does not cluster with either group.

FIG. 4 shows a heat map which demonstrates synergy found betweenLapatinib and the AKT inhibitor GSK690693 is dependent upon PI3K pathwaystatus. Combination index values were calculated across all nine dosesof drug and transformed to take into account significance (upper 95%confidence interval less than 1). Significant synergy is denoted aslight gray indicates significant synergy, black indicates additivity/nosignificant interaction, and dark gray indicates antagonismin the heatmap. Clustering of the data revealed that all the lines with similarsynergistic responses had PI3K pathway mutations (cells with mutationsin the PI3K pathway are denoted in red or blue). In contrast, all buttwo (BT474, MDAMB453) of the non-synergistic lines were wild-type forPI3K.

FIG. 5 shows a heatmap which demonstrates synergy found betweenLapatinib and the AKT inhibitor GSK2141795 is dependent upon PI3Kpathway status. Combination index values were calculated across all ninedoses of drug and transformed to take into account significance (upper95% confidence interval less than 1). Significant synergy is denoted aslight gray indicates significant synergy, black indicates additivity/nosignificant interaction, and dark gray indicates antagonism in the heatmap. Clustering of the data revealed that all the lines with similarsynergistic responses had PI3K pathway mutations except one (cells withmutations in the PI3K pathway are denoted in red or blue). In contrast,all but two (BT474, HCC1569) of the non-synergistic lines were wild-typefor PI3K.

FIG. 6 shows graphs showing that introduction of a PIK3CA mutationresults in synergy in previously non-synergistic HER2+ cell line. A)Dose response of EFM192A cells (wild-type PI3K pathway) with laptinib(square), AKTi (GSK690693, triangle), or a combination of the two(diamond) does not result in significant synergism. B) Introduction ofan H1047R PIK3CA mutation into EFM192A results in significant synergywhen treated with the same drug combination as in A (points withsignificant synergism are indicated with asterisks).

Table 1. Average GI50 values and combination indices at IC50 values tolapatinib, AKTi, or a combination of the two in HER2 positive cell lineswith known PI3K mutation (either PIK3CA or PTEN; N=7) compared to thosewith known WT PI3K (N=5). Cell lines highlighted in bold (HCC202;MDAMB361; HCC1954; SUM190PT) show strongest synergistic interactionbetween Lapatinib and AKT.

Table 2. List of cell lines used in the Examples (note: MDAMB175 is notHER2 amplified and so was excluded from the synergy experiments).

DETAILED DESCRIPTION

The present invention provides a new combination of targeted therapeuticagents that should have clinical efficacy for the treatment of HER2positive breast cancer. The present invention also provides as geneticmarkers mutant PIK3CA and SASH1 which are herein shown to be predictiveof patients most likely to respond to this combination.

We have found and shown that a combination of Lapatinib with an AKT geneinhibitor is synergistic in HER2 positive patients who also havemutations in the PIK3CA gene and the PTEN gene (see FIG. 2). We alsofound that the combination could be antagonistic at high doses inpatients with wild-type PIK3CA, indicating that careful patientselection will be required to ensure optimal patient populations fortreatment with this drug combination. We also found that high levels ofSASH1 gene expression predict response (and are correlated with PIK3CAmutation status). Thus, it represents an important advance byidentifying both a new combination of targeted therapeutic agents withpotential clinical efficacy and biomarkers that predict which patientsare most likely to respond to this combination. This should result inimproved treatment options for patients with HER2 positive cancer.

The cancer patient is either patient who is known to be ERBB2-positive,i.e., HER2-positive, and overexpresses the ERBB2 protein also known asthe HER2 protein, or it is not known whether patient is ERBB2-positiveor not. When the patient is not known whether to be ERBB2-positive ornot, the ERBB2 status of the patient is to be determined.

The present methods describe the measurement and detection of expressionlevels of a gene as measured from a sample from a patient that comprisesessentially a cancer cell or cancer tissue of a cancer tumor. Suchmethods for obtaining such samples are well known to those skilled inthe art. When the cancer is breast cancer, the expression level of agene is measured from a sample from the patient that comprisesessentially a breast cancer cell or breast cancer tissue of a breastcancer tumor.

Methods for detection of expression levels of a gene can be carried outusing known methods in the art including but not limited to, fluorescentin situ hybridization (FISH), immunohistochemical analysis, comparativegenomic hybridization, PCR methods including real-time and quantitativePCR, and other sequencing and analysis methods. The expression level ofthe gene in question can be measured by measuring the amount or numberof molecules of mRNA or transcript in a cell. The measuring can comprisedirectly measuring the mRNA or transcript obtained from a cell, ormeasuring the cDNA obtained from an mRNA preparation thereof. Suchmethods of extracting the mRNA or transcript from a cell, or preparingthe cDNA thereof are well known to those skilled in the art. In otherembodiments, the expression level of a gene can be measured by measuringor detecting the amount of protein or polypeptide expressed, such asmeasuring the amount of antibody that specifically binds to the proteinin a dot blot or Western blot. The proteins described in the presentinvention can be overexpressed and purified or isolated to homogeneityand antibodies raised that specifically bind to each protein. Suchmethods are well known to those skilled in the art.

Comparison of the detected expression level of a gene in a patientsample is often compared to the expression levels detected in a normaltissue sample or a reference expression level. In some embodiments, thereference expression level can be the average or normalized expressionlevel of the gene in a panel of normal cell lines or cancer cell lines.

In some embodiments, the method comprises measuring the expression levelof ERBB2 of the patients in order to determine whether the patient is anERBB2-positive patient. The expression level of a gene encoding ERBB2can be measured using an oligonucleotide derived from the mouse v-erb-b2erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastomaderived oncogene homolog (avian) (Erbb2), mRNA sequence of GenBankAccession No. NM_(—)001003817.1 GI:54873609, hereby incorporated byreference and shown as SEQ ID NO: 1:

   1 cgaggaagtg cggcgtgaag ttgtggagct gagattgccc gccgctgggg acccggagcc  61 caggagcgcc ccttcccagg cggccccttc cggcgccgcg cctgtgcctg ccctcgccgc 121 gccccgcgcc cgcagcctgg tccagcctga gccatggggc cggagccgca gtgatcatca 181 tggagctggc ggcctggtgc cgttgggggt tcctcctcgc cctcctgtcc cccggagccg 241 cgggtaccca agtgtgtacc ggtaccgaca tgaagttgcg actccctgcc agtcctgaga 301 cccacctgga catgcttcgc cacctctacc agggctgtca ggtggtgcag ggcaatttgg 361 agcttaccta cctgcccgcc aatgccagcc tctcattcct gcaggacatc caggaagtcc 421 agggatacat gctcatcgct cacaaccgag tgaaacacgt cccactgcag aggttgcgca 481 tcgtgagagg gactcagctc tttgaggaca agtatgccct ggctgtgcta gacaaccgag 541 accctttgga caacgtcacc accgccgccc caggcagaac cccagaaggg ctgcgggagc 601 tgcagcttcg aagtctcaca gagatcttga agggaggagt tttgatccgt gggaaccctc 661 agctctgcta ccaggacatg gttttgtgga aggatgtcct ccgtaagaat aaccagctgg 721 ctcctgtcga catggacacc aatcgttccc gggcctgtcc accttgtgcc ccaacctgca 781 aagacaatca ctgttggggt gagagtcctg aagactgtca gatcttgact ggcaccatct 841 gtactagtgg ctgtgcccgg tgcaagggcc ggctgcccac tgactgttgc catgagcagt 901 gtgctgcagg ctgcacgggt cccaagcatt ctgactgcct ggcctgcctc cacttcaatc 961 atagtggtat ctgtgagctg cactgcccgg ccctcatcac ctacaacaca gacaccttcg1021 agtccatgct caaccctgag ggtcgctaca cctttggtgc cagctgtgtg accacctgcc1081 cctacaacta cctctccacg gaagtgggat cctgcactct ggtctgtccc ccgaacaacc1141 aagaggtcac agctgaggac ggaacacagc ggtgtgagaa atgcagcaag ccctgtgctg1201 gagtatgcta tggtctgggc atggagcacc tccgaggggc gagggccatc accagtgaca1261 atatccagga gtttgctggc tgcaagaaga tctttgggag cctggcattt ttgccggaga1321 gctttgatgg gaacccctcc tccggcgttg ccccactgaa gccagagcat ctccaagtgt1381 tcgaaaccct ggaggagatc acaggttacc tatacatttc agcatggcca gagagcttcc1441 aagacctcag tgtcttccag aaccttcggg tcattcgggg acggattctc catgatggtg1501 cttactcatt gacgttgcaa ggcctgggga ttcactcact ggggctacgc tcactgcggg1561 agctgggcag tggattggct ctcattcacc gcaacaccca tctctgcttt gtaaacactg1621 taccttggga ccagctcttc cggaacccgc accaggccct actccacagt gggaaccggc1681 cagaagaggc atgtggtctt gagggcttgg tctgtaactc actgtgtgcc cgtgggcact1741 gctgggggcc agggcccacc cagtgtgtca actgcagtca gttcctccgg ggccaggagt1801 gtgtggagga gtgccgagta tggaaggggc tccccaggga gtatgtgagg ggcaagcact1861 gtctgccatg ccaccccgag tgtcagcctc aaaacagctc ggagacctgc tatggatcgg1921 aggctgacca gtgtgaggct tgtgcccact acaaggactc atcttcctgt gtggctcgct1981 gccccagtgg tgtgaagcca gacctctcct acatgcctat ctggaagtac ccggatgagg2041 agggcatatg tcagccatgc cccatcaact gcacccactc atgtgtggac ctggacgaac2101 gaggctgccc agcagagcag agagccagcc cagtgacatt catcattgca actgtggtgg2161 gcgtcctgtt gttcctgatc atagtggtgg tcattggaat cctaatcaaa cgaaggcgac2221 agaagatccg gaagtatacc atgcgtaggc tgctgcagga gaccgagctg gtggagccgc2281 tgacgcccag tggagctgtg cccaaccagg ctcagatgcg gatcctaaag gagacagagc2341 taaggaagct gaaggtgctt gggtcaggag ccttcggcac tgtctacaag ggcatctgga2401 tcccagatgg ggagaacgtg aaaatccccg tggccatcaa ggtgttgagg gaaaacacat2461 ctcctaaagc taacaaagaa atcctagatg aagcgtacgt catggctggt gtgggttctc2521 catatgtgtc ccgcctcctg ggcatctgcc tgacatccac agtgcagctg gtgacacagc2581 ttatgcccta tggctgcctt ctggaccatg tccgagaaca ccgaggtcgc ttaggctccc2641 aggacctgct caactggtgt gttcagattg ccaaggggat gagctacctg gaggaagttc2701 ggcttgttca cagggaccta gctgcccgaa acgtgctagt caagagtccc aaccacgtca2761 agattaccga cttcgggctg gcacggctgc tggacattga tgagactgaa taccatgcag2821 atgggggcaa ggtgcccatc aagtggatgg cattggaatc tattctcaga cgccggttca2881 cccatcagag tgatgtgtgg agctatggtg tgactgtgtg ggagctgatg acctttgggg2941 ccaaacctta cgatgggatc ccagctcggg agatccctga tttgctggag aagggagaac3001 gcctacctca gcctccaatc tgcaccatcg acgtctacat gatcatggtc aaatgttgga3061 tgattgactc cgaatgtcgc ccgagattcc gggagttggt atcagaattc tcccgtatgg3121 caagggaccc ccagcgcttt gtggtcatcc agaacgagga cttaggcccc tccagcccca3181 tggacagcac cttctaccgt tcactgctgg aggatgatga catgggggag ctggtcgatg3241 ctgaagagta cctggtaccc cagcagggat tcttctcccc agaccctgcc ctaggtactg3301 ggagcacagc ccaccgcaga caccgcagct cgtcggccag gagtggcggt ggtgagctga3361 cactgggcct ggagccctcg gaagaagagc cccccagatc tccactggct ccctccgaag3421 gggctggctc cgatgtgttt gatggtgacc tggcagtggg ggtaaccaaa ggactgcaga3481 gcctctctcc acatgacctc agccctctac agcggtacag tgaggatccc acattacctc3541 tgccccccga gactgatggc tacgttgctc ccctggcctg cagcccccag cccgagtatg3601 tgaaccagcc agaggttcgg cctcagtctc ccttgacccc agagggtcct ccgcctccca3661 tccgacctgc tggtgctact ctagaaagac ccaagactct ctctcctggg aaaaatgggg3721 ttgtcaaaga cgtttttgcc tttgggggtg ctgtggagaa ccctgaatac ttagcaccca3781 gagcaggcac tgcctctcag ccccaccctt ctcctgcctt cagcccagcc tttgacaacc3841 tctattactg ggaccagaac tcatcggagc agggtcctcc accaagtacc tttgaaggga3901 cccccactgc agagaaccct gagtacctag gcctggatgt gccagtatga ggtcacatgt3961 gcagacatcc tctgtcttca gagtggggaa ggaaggccta acttgtggtc tccatcgccc4021 gccacaaagc agggagaagg tcctctggcc acatgacatc cagggcagcc ggctatgcca4081 ggaacgtgcc ctgaggaacc tcgctcgatg cttcaatcct gagtggttaa gagggccccg4141 cctggccgga agagacagca cactgttcag ccccagagga ttacagaccc tgactgccct4201 gacagactgt agggtccagt gggtattcct tacctggcct ggctctcttg gttctgaaga4261 ctgagggaag ctcagcctgc aagggaggag gccccaggtg aatatcctgg gagcaggaca4321 ccccactagg actgaggcac gtgcatccca agagggggac agcacttgca cccagactgg4381 tctttgtaca gagtttattt tgttctgttt ttacttttgt tttttgtttt ttttttaaag4441 atgaaataag gatacagtgg gagagtgggt gttatatgaa agtcgggggg tgctgtcccc4501 tttctccatt tgcaatgaga tttgtaaaat aactggaccc cagcctatgt ctgagagtgg4561 tcccgggccg ggtcaaaccg tattgctcat ctgacacaca gctcctcctg gagtgagtgt4621 gtagagatct tccaaaagtt tgagacaatt tggctttggg cttgagggac tggggagtta4681 ggattccttc tgaaggccct ttggcaacag ggtcattctc cgttggacac actcatacca4741 aggctacccc cagaatactc cgttggacac actcattcca aggctacccc cagaatgaag4801 tcctgtcctc ccagtgggag aggggagctt gtggagagca ttgccatgtg acttgttttc4861 cttgccttag aaagaagtat ccatccagga aaaccccacc cactaggtgt tagtcccacc4921 cactaggtgt tagcagggcc agactgacct gtgtgccccc cgcacaggct ggacataaac4981 acacgccagt tgacacaa

The expression level of a gene encoding ERBB2 can also be measured usingan oligonucleotide derived from the human nucleotide sequence of GenBankAccession No. NM_(—)004448.2 GI:54792095, Homo sapiens v-erb-b2erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastomaderived oncogene homolog (avian) (ERBB2), transcript variant 1, mRNA,hereby incorporated by reference and shown as SEQ ID NO: 2:

   1 ggaggaggtg gaggaggagg gctgcttgag gaagtataag aatgaagttg tgaagctgag  61 attcccctcc attgggaccg gagaaaccag gggagccccc cgggcagccg cgcgcccctt 121 cccacggggc cctttactgc gccgcgcgcc cggcccccac ccctcgcagc accccgcgcc 181 ccgcgccctc ccagccgggt ccagccggag ccatggggcc ggagccgcag tgagcaccat 241 ggagctggcg gccttgtgcc gctgggggct cctcctcgcc ctcttgcccc ccggagccgc 301 gagcacccaa gtgtgcaccg gcacagacat gaagctgcgg ctccctgcca gtcccgagac 361 ccacctggac atgctccgcc acctctacca gggctgccag gtggtgcagg gaaacctgga 421 actcacctac ctgcccacca atgccagcct gtccttcctg caggatatcc aggaggtgca 481 gggctacgtg ctcatcgctc acaaccaagt gaggcaggtc ccactgcaga ggctgcggat 541 tgtgcgaggc acccagctct ttgaggacaa ctatgccctg gccgtgctag acaatggaga 601 cccgctgaac aataccaccc ctgtcacagg ggcctcccca ggaggcctgc gggagctgca 661 gcttcgaagc ctcacagaga tcttgaaagg aggggtcttg atccagcgga acccccagct 721 ctgctaccag gacacgattt tgtggaagga catcttccac aagaacaacc agctggctct 781 cacactgata gacaccaacc gctctcgggc ctgccacccc tgttctccga tgtgtaaggg 841 ctcccgctgc tggggagaga gttctgagga ttgtcagagc ctgacgcgca ctgtctgtgc 901 cggtggctgt gcccgctgca aggggccact gcccactgac tgctgccatg agcagtgtgc 961 tgccggctgc acgggcccca agcactctga ctgcctggcc tgcctccact tcaaccacag1021 tggcatctgt gagctgcact gcccagccct ggtcacctac aacacagaca cgtttgagtc1081 catgcccaat cccgagggcc ggtatacatt cggcgccagc tgtgtgactg cctgtcccta1141 caactacctt tctacggacg tgggatcctg caccctcgtc tgccccctgc acaaccaaga1201 ggtgacagca gaggatggaa cacagcggtg tgagaagtgc agcaagccct gtgcccgagt1261 gtgctatggt ctgggcatgg agcacttgcg agaggtgagg gcagttacca gtgccaatat1321 ccaggagttt gctggctgca agaagatctt tgggagcctg gcatttctgc cggagagctt1381 tgatggggac ccagcctcca acactgcccc gctccagcca gagcagctcc aagtgtttga1441 gactctggaa gagatcacag gttacctata catctcagca tggccggaca gcctgcctga1501 cctcagcgtc ttccagaacc tgcaagtaat ccggggacga attctgcaca atggcgccta1561 ctcgctgacc ctgcaagggc tgggcatcag ctggctgggg ctgcgctcac tgagggaact1621 gggcagtgga ctggccctca tccaccataa cacccacctc tgcttcgtgc acacggtgcc1681 ctgggaccag ctctttcgga acccgcacca agctctgctc cacactgcca accggccaga1741 ggacgagtgt gtgggcgagg gcctggcctg ccaccagctg tgcgcccgag ggcactgctg1801 gggtccaggg cccacccagt gtgtcaactg cagccagttc cttcggggcc aggagtgcgt1861 ggaggaatgc cgagtactgc aggggctccc cagggagtat gtgaatgcca ggcactgttt1921 gccgtgccac cctgagtgtc agccccagaa tggctcagtg acctgttttg gaccggaggc1981 tgaccagtgt gtggcctgtg cccactataa ggaccctccc ttctgcgtgg cccgctgccc2041 cagcggtgtg aaacctgacc tctcctacat gcccatctgg aagtttccag atgaggaggg2101 cgcatgccag ccttgcccca tcaactgcac ccactcctgt gtggacctgg atgacaaggg2161 ctgccccgcc gagcagagag ccagccctct gacgtccatc atctctgcgg tggttggcat2221 tctgctggtc gtggtcttgg gggtggtctt tgggatcctc atcaagcgac ggcagcagaa2281 gatccggaag tacacgatgc ggagactgct gcaggaaacg gagctggtgg agccgctgac2341 acctagcgga gcgatgccca accaggcgca gatgcggatc ctgaaagaga cggagctgag2401 gaaggtgaag gtgcttggat ctggcgcttt tggcacagtc tacaagggca tctggatccc2461 tgatggggag aatgtgaaaa ttccagtggc catcaaagtg ttgagggaaa acacatcccc2521 caaagccaac aaagaaatct tagacgaagc atacgtgatg gctggtgtgg gctccccata2581 tgtctcccgc cttctgggca tctgcctgac atccacggtg cagctggtga cacagcttat2641 gccctatggc tgcctcttag accatgtccg ggaaaaccgc ggacgcctgg gctcccagga2701 cctgctgaac tggtgtatgc agattgccaa ggggatgagc tacctggagg atgtgcggct2761 cgtacacagg gacttggccg ctcggaacgt gctggtcaag agtcccaacc atgtcaaaat2821 tacagacttc gggctggctc ggctgctgga cattgacgag acagagtacc atgcagatgg2881 gggcaaggtg cccatcaagt ggatggcgct ggagtccatt ctccgccggc ggttcaccca2941 ccagagtgat gtgtggagtt atggtgtgac tgtgtgggag ctgatgactt ttggggccaa3001 accttacgat gggatcccag cccgggagat ccctgacctg ctggaaaagg gggagcggct3061 gccccagccc cccatctgca ccattgatgt ctacatgatc atggtcaaat gttggatgat3121 tgactctgaa tgtcggccaa gattccggga gttggtgtct gaattctccc gcatggccag3181 ggacccccag cgctttgtgg tcatccagaa tgaggacttg ggcccagcca gtcccttgga3241 cagcaccttc taccgctcac tgctggagga cgatgacatg ggggacctgg tggatgctga3301 ggagtatctg gtaccccagc agggcttctt ctgtccagac cctgccccgg gcgctggggg3361 catggtccac cacaggcacc gcagctcatc taccaggagt ggcggtgggg acctgacact3421 agggctggag ccctctgaag aggaggcccc caggtctcca ctggcaccct ccgaaggggc3481 tggctccgat gtatttgatg gtgacctggg aatgggggca gccaaggggc tgcaaagcct3541 ccccacacat gaccccagcc ctctacagcg gtacagtgag gaccccacag tacccctgcc3601 ctctgagact gatggctacg ttgcccccct gacctgcagc ccccagcctg aatatgtgaa3661 ccagccagat gttcggcccc agcccccttc gccccgagag ggccctctgc ctgctgcccg3721 acctgctggt gccactctgg aaaggcccaa gactctctcc ccagggaaga atggggtcgt3781 caaagacgtt tttgcctttg ggggtgccgt ggagaacccc gagtacttga caccccaggg3841 aggagctgcc cctcagcccc accctcctcc tgccttcagc ccagccttcg acaacctcta3901 ttactgggac caggacccac cagagcgggg ggctccaccc agcaccttca aagggacacc3961 tacggcagag aacccagagt acctgggtct ggacgtgcca gtgtgaacca gaaggccaag4021 tccgcagaag ccctgatgtg tcctcaggga gcagggaagg cctgacttct gctggcatca4081 agaggtggga gggccctccg accacttcca ggggaacctg ccatgccagg aacctgtcct4141 aaggaacctt ccttcctgct tgagttccca gatggctgga aggggtccag cctcgttgga4201 agaggaacag cactggggag tctttgtgga ttctgaggcc ctgcccaatg agactctagg4261 gtccagtgga tgccacagcc cagcttggcc ctttccttcc agatcctggg tactgaaagc4321 cttagggaag ctggcctgag aggggaagcg gccctaaggg agtgtctaag aacaaaagcg4381 acccattcag agactgtccc tgaaacctag tactgccccc catgaggaag gaacagcaat4441 ggtgtcagta tccaggcttt gtacagagtg cttttctgtt tagtttttac tttttttgtt4501 ttgttttttt aaagatgaaa taaagaccca gggggagaat gggtgttgta tggggaggca4561 agtgtggggg gtccttctcc acacccactt tgtccatttg caaatatatt ttggaaaaca4621 gcta

Methods of assaying for ERBB2 or HER2 protein overexpression includemethods that utilize immunohistochemistry (1HC) and methods that utilizefluorescence in situ hybridization (FISH). A commercially available IHCtest is DAKO HercepTest® (DAKO Corp., Carpinteria, Calif.). Patientsamples having an IHC staining score of 0-1, 2 is normal, and scores of2+ may be borerderline, while results of 2, 3+ are scored as positivefor multiple copies of HER2 (HER2 positive).

A commercially available FISH test is PathVysion® (Vysis Inc., DownersGrove, Ill.). The HER2 genomic copy number of a patient sample isdetermined using FISH. Generally if a sample is found to have 3.6 ormore copies of HER2 (normal=2 copies), the patient is determined to beHER2 positive.

While many HER2-positive patients suffer from metastatic breast cancer,a patient's HER2 status can also be determined in relation to othertypes of cancers including but not limited to epithelial cancers such aspancreatic, lung, cervical, ovarian, prostate, non-small cell lungcarcinomas, melanomas, squamous cell cancers, etc. It is contemplatedthat the present methods described herein may find use in prognosis andpredicting patient response to certain combination therapies that may beused in various cancer treatments for multiple types of cancers so longas the patient criteria described herein is present as identifying apatient suitable for such combination therapy.

In one embodiment a method for identifying a HER2-positive cancerpatient suitable for treatment with a 4-anilinoquinazoline kinaseinhibitor and an AKT inhibitor, comprising: (a) obtaining the sequenceof the PIK3CA gene from a sample from a patient; and (b) identifying anymutations in the PIK3CA gene in a sample from the patient as compared tothe wild-type sequence found in SEQ ID NO:1, wherein a mutation in thePIK3CA gene indicates the patient is suitable for treatment with the4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.

The PIK3CA gene is identified as Homo sapiens phosphoinositide-3-kinase,catalytic, alpha polypeptide (PIK3CA) on chromosome 3, and the wild-typePIK3CA gene sequence is found at GenBankAccession No. NG 012113.1GI:237858742, hereby incorporated by reference and identified as SEQ IDNO:11.

The PIK3CA mRNA sequence is found at NM_(—)006218.2 GI:54792081, herebyincorporated by reference, and shown here as SEQ ID NO:3.

   1 tctccctcgg cgccgccgcc gccgcccgcg gggctgggac ccgatgcggt tagagccgcg  61 gagcctggaa gagccccgag cgtttctgct ttgggacaac catacatcta attccttaaa 121 gtagttttat atgtaaaact tgcaaagaat cagaacaatg cctccacgac catcatcagg 181 tgaactgtgg ggcatccact tgatgccccc aagaatccta gtagaatgtt tactaccaaa 241 tggaatgata gtgactttag aatgcctccg tgaggctaca ttaataacca taaagcatga 301 actatttaaa gaagcaagaa aataccccct ccatcaactt cttcaagatg aatcttctta 361 cattttcgta agtgttactc aagaagcaga aagggaagaa ttttttgatg aaacaagacg 421 actttgtgac cttcggcttt ttcaaccctt tttaaaagta attgaaccag taggcaaccg 481 tgaagaaaag atcctcaatc gagaaattgg ttttgctatc ggcatgccag tgtgtgaatt 541 tgatatggtt aaagatccag aagtacagga cttccgaaga aatattctga acgtttgtaa 601 agaagctgtg gatcttaggg acctcaattc acctcatagt agagcaatgt atgtctatcc 661 tccaaatgta gaatcttcac cagaattgcc aaagcacata tataataaat tagataaagg 721 gcaaataata gtggtgatct gggtaatagt ttctccaaat aatgacaagc agaagtatac 781 tctgaaaatc aaccatgact gtgtaccaga acaagtaatt gctgaagcaa tcaggaaaaa 841 aactcgaagt atgttgctat cctctgaaca actaaaactc tgtgttttag aatatcaggg 901 caagtatatt ttaaaagtgt gtggatgtga tgaatacttc ctagaaaaat atcctctgag 961 tcagtataag tatataagaa gctgtataat gcttgggagg atgcccaatt tgatgttgat1021 ggctaaagaa agcctttatt ctcaactgcc aatggactgt tttacaatgc catcttattc1081 cagacgcatt tccacagcta caccatatat gaatggagaa acatctacaa aatccctttg1141 ggttataaat agtgcactca gaataaaaat tctttgtgca acctacgtga atgtaaatat1201 tcgagacatt gataagatct atgttcgaac aggtatctac catggaggag aacccttatg1261 tgacaatgtg aacactcaaa gagtaccttg ttccaatccc aggtggaatg aatggctgaa1321 ttatgatata tacattcctg atcttcctcg tgctgctcga ctttgccttt ccatttgctc1381 tgttaaaggc cgaaagggtg ctaaagagga acactgtcca ttggcatggg gaaatataaa1441 cttgtttgat tacacagaca ctctagtatc tggaaaaatg gctttgaatc tttggccagt1501 acctcatgga ttagaagatt tgctgaaccc tattggtgtt actggatcaa atccaaataa1561 agaaactcca tgcttagagt tggagtttga ctggttcagc agtgtggtaa agttcccaga1621 tatgtcagtg attgaagagc atgccaattg gtctgtatcc cgagaagcag gatttagcta1681 ttcccacgca ggactgagta acagactagc tagagacaat gaattaaggg aaaatgacaa1741 agaacagctc aaagcaattt ctacacgaga tcctctctct gaaatcactg agcaggagaa1801 agattttcta tggagtcaca gacactattg tgtaactatc cccgaaattc tacccaaatt1861 gcttctgtct gttaaatgga attctagaga tgaagtagcc cagatgtatt gcttggtaaa1921 agattggcct ccaatcaaac ctgaacaggc tatggaactt ctggactgta attacccaga1981 tcctatggtt cgaggttttg ctgttcggtg cttggaaaaa tatttaacag atgacaaact2041 ttctcagtat ttaattcagc tagtacaggt cctaaaatat gaacaatatt tggataactt2101 gcttgtgaga tttttactga agaaagcatt gactaatcaa aggattgggc actttttctt2161 ttggcattta aaatctgaga tgcacaataa aacagttagc cagaggtttg gcctgctttt2221 ggagtcctat tgtcgtgcat gtgggatgta tttgaagcac ctgaataggc aagtcgaggc2281 aatggaaaag ctcattaact taactgacat tctcaaacag gagaagaagg atgaaacaca2341 aaaggtacag atgaagtttt tagttgagca aatgaggcga ccagatttca tggatgctct2401 acagggcttt ctgtctcctc taaaccctgc tcatcaacta ggaaacctca ggcttgaaga2461 gtgtcgaatt atgtcctctg caaaaaggcc actgtggttg aattgggaga acccagacat2521 catgtcagag ttactgtttc agaacaatga gatcatcttt aaaaatgggg atgatttacg2581 gcaagatatg ctaacacttc aaattattcg tattatggaa aatatctggc aaaatcaagg2641 tcttgatctt cgaatgttac cttatggttg tctgtcaatc ggtgactgtg tgggacttat2701 tgaggtggtg cgaaattctc acactattat gcaaattcag tgcaaaggcg gcttgaaagg2761 tgcactgcag ttcaacagcc acacactaca tcagtggctc aaagacaaga acaaaggaga2821 aatatatgat gcagccattg acctgtttac acgttcatgt gctggatact gtgtagctac2881 cttcattttg ggaattggag atcgtcacaa tagtaacatc atggtgaaag acgatggaca2941 actgtttcat atagattttg gacacttttt ggatcacaag aagaaaaaat ttggttataa3001 acgagaacgt gtgccatttg ttttgacaca ggatttctta atagtgatta gtaaaggagc3061 ccaagaatgc acaaagacaa gagaatttga gaggtttcag gagatgtgtt acaaggctta3121 tctagctatt cgacagcatg ccaatctctt cataaatctt ttctcaatga tgcttggctc3181 tggaatgcca gaactacaat cttttgatga cattgcatac attcgaaaga ccctagcctt3241 agataaaact gagcaagagg ctttggagta tttcatgaaa caaatgaatg atgcacatca3301 tggtggctgg acaacaaaaa tggattggat cttccacaca attaaacagc atgcattgaa3361 ctgaaaagat aactgagaaa atgaaagctc actctggatt ccacactgca ctgttaataa3421 ctctcagcag gcaaagaccg attgcatagg aattgcacaa tccatgaaca gcattagaat3481 ttacagcaag aacagaaata aaatactata taatttaaat aatgtaaacg caaacagggt3541 ttgatagcac ttaaactagt tcatttcaaa attaagcttt agaataatgc gcaatttcat3601 gttatgcctt aagtccaaaa aggtaaactt tgaagattgt ttgtatcttt ttttaaaaaa3661 caaaacaaaa caaaaatccc caaaatatat agaaatgatg gagaaggaaa aaaaaaaaaa3721 aaaa //

The PIK3CA mRNA is expressed as thephosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alphaisoform [Homo sapiens](PI3_K or PIK3CA) protein having GenBank AccessionNo. NP 006209.2 GI:54792082, hereby incorporated by reference, and shownhere as SEQ ID NO:4.

   1 mpprpssgel wgihlmppri lvecllpngm ivtleclrea tlitikhelf kearkyplhq  61 llqdessyif vsvtqeaere effdetrrlc dlrlfqpflk viepvgnree kilnreigfa 121 igmpvcefdm vkdpevqdfr rnilnvckea vdlrdlnsph sramyvyppn vesspelpkh 181 iynkldkgqi ivviwvivsp nndkqkytlk inhdcvpeqv iaeairkktr smllsseqlk 241 lcvleyqgky ilkvcgcdey flekyplsqy kyirscimlg rmpnlmlmak eslysqlpmd 301 cftmpsysrr istatpymng etstkslwvi nsalrikilc atyvnvnird idkiyvrtgi 361 yhggeplcdn vntqrvpcsn prwnewlnyd iyipdlpraa rlclsicsvk grkgakeehc 421 plawgninlf dytdtivsgk malnlwpvph gledllnpig vtgsnpnket pclelefdwf 481 ssvvkfpdms vieehanwsv sreagfsysh aglsnrlard nelrendkeq lkaistrdpl 541 seiteqekdf lwshrhycvt ipeilpklll svkwnsrdev aqmyclvkdw ppikpeqame 601 lldcnypdpm vrgfavrcle kyltddklsq yliqlvqvlk yeqyldnllv rfllkkaltn 661 qrighfffwh lksemhnktv sqrfgllles ycracgmylk hlnrqveame klinltdilk 721 qekkdetqkv qmkflveqmr rpdfmdalqg flspinpahq lgnlrleecr imssakrplw 781 lnwenpdims ellfqnneii fkngddlrqd mltlqiirim eniwqnqgld lrmlpygcls 841 igdcvgliev vrnshtimqi qckgglkgal qfnshtlhqw lkdknkgeiy daaidlftrs 901 cagycvatfi lgigdrhnsn imvkddgqlf hidfghfldh kkkkfgykre rvpfvltqdf 961 liviskgaqe ctktreferf qemcykayla irqhanlfin lfsmmlgsgm pelqsfddia1021 yirktlaldk teqealeyfm kqmndahhgg wttkmdwifh tikqhaln //

Missense mutations are most commonly found in two hotspot locations inPIK3CA, occurring in either the helical domain (amino acids 542 545) orthe kinase domain (amino acids 1047 and 1049). See Saal et al. CancerResearch 65, 2554-2559, Apr. 1, 2005, “PIK3CA Mutations Correlate withHormone Receptors, Node Metastasis, and ERBB2, and Are MutuallyExclusive with PTEN Loss in Human Breast Carcinoma,” hereby incorporatedby reference. PIK3CA missense mutations detected by Saal et al. andothers have found the following substitutions: E542K, E545K, H1047R,H1047L, H1047Y and G1049R. Furthermore, tumors with both PIK3CA copynumber gain and mutation had moderate levels of gain and were enrichedfor E545K and other non-H1047R mutations.

Table 1 shows the various mutations found in the cell lines tested andinclude: E545K, H1047R, K111N, E307K. Cell lines having these mutationswere found to have synergistic response. Either mutation is thought tobe sufficient to activate the PI3K pathway, and both of these mutationshave been shown to be transforming in vitro. Mutation of the PIK3CA geneis one of the most common mutations found in breast cancer, withmutation rates exceeding 20%. While cell lines from patients with the542/545 mutations appear to be most resistant and those with 1047mutation appear to be most sensitive to lapatinib as a monotherapy, bothtypes appear to have comparable synergistic responses to the combinationof lapatinib plus an AKT inhibitor.

Thus, in one embodiment, a biopsy, tissue or fluid sample is obtainedfrom a patient and the mutation status of a patient at the PIK3CA geneis obtained by methods known and used the in the art. The two mainPIK3CA mutation sites: aa 542-545 (E542K, E545K) and aa 1047 (H1047R,H1047L) correspond to PIK3CA mRNA sequence sites of 1624 bp, 1633, 1634and 3140 bp. For example, in one embodiment, the patient's PIK3CA geneor mRNA is amplified and sequenced to identify any mutations in thePIK3CA gene. It may be preferable to use primers within the PIK3CA geneor mRNA sequence that are near the two hotspot locations for PIK3CAmutation to use to amplify those regions and then the resulting PCRproducts sequenced.

Other methods that can identify mutations in the candidate gene can beused. In one embodiment, the PIK3CA mutation in the PIK3CA proteinexpressed in the patient's tissue is identified. For example,immunohistochemical analysis by utilizing an antibody that specificallyidentifies an E545K or an H1047R mutation can be performed.

Two cell lines having a mutation in the PTEN gene were found. MDAMB453features a E307 K mutation in PTEN and showed synergistic response toboth lapatinib and the AKT inhibitor. Another cell line HCC1569 with aPTEN mutation showed antagonistic response to the combination therapy.It may be useful to identify the mutation status of a patient at thePTEN gene.

The PTEN gene was identified as a tumor suppressor that is mutated in alarge number of cancers at high frequency. The protein encoded this geneis a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It containsa tensin like domain as well as a catalytic domain similar to that ofthe dual specificity protein tyrosine phosphatases. Unlike most of theprotein tyrosine phosphatases, this protein preferentiallydephosphorylates phosphoinositide substrates. It negatively regulatesintracellular levels of phosphatidylinositol-3,4,5-trisphosphate incells and functions as a tumor suppressor by negatively regulatingAKT/PKB signaling pathway. The expression level or the mutation statuscan be evaluated using primers or oligonucleotides derived from PTENmRNA sequence found at GenBank Accession No. NM_(—)000314.4GI:110224474, hereby incorporated by reference and shown here as SEQ IDNO:5:

   1 cctcccctcg cccggcgcgg tcccgtccgc ctctcgctcg cctcccgcct cccctcggtc  61 ttccgaggcg cccgggctcc cggcgcggcg gcggaggggg cgggcaggcc ggcgggcggt 121 gatgtggcgg gactctttat gcgctgcggc aggatacgcg ctcggcgctg ggacgcgact 181 gcgctcagtt ctctcctctc ggaagctgca gccatgatgg aagtttgaga gttgagccgc 241 tgtgaggcga ggccgggctc aggcgaggga gatgagagac ggcggcggcc gcggcccgga 301 gcccctctca gcgcctgtga gcagccgcgg gggcagcgcc ctcggggagc cggccggcct 361 gcggcggcgg cagcggcggc gtttctcgcc tcctcttcgt cttttctaac cgtgcagcct 421 cttcctcggc ttctcctgaa agggaaggtg gaagccgtgg gctcgggcgg gagccggctg 481 aggcgcggcg gcggcggcgg cacctcccgc tcctggagcg ggggggagaa gcggcggcgg 541 cggcggccgc ggcggctgca gctccaggga gggggtctga gtcgcctgtc accatttcca 601 gggctgggaa cgccggagag ttggtctctc cccttctact gcctccaaca cggcggcggc 661 ggcggcggca catccaggga cccgggccgg ttttaaacct cccgtccgcc gccgccgcac 721 cccccgtggc ccgggctccg gaggccgccg gcggaggcag ccgttcggag gattattcgt 781 cttctcccca ttccgctgcc gccgctgcca ggcctctggc tgctgaggag aagcaggccc 841 agtcgctgca accatccagc agccgccgca gcagccatta cccggctgcg gtccagagcc 901 aagcggcggc agagcgaggg gcatcagcta ccgccaagtc cagagccatt tccatcctgc 961 agaagaagcc ccgccaccag cagcttctgc catctctctc ctcctttttc ttcagccaca1021 ggctcccaga catgacagcc atcatcaaag agatcgttag cagaaacaaa aggagatatc1081 aagaggatgg attcgactta gacttgacct atatttatcc aaacattatt gctatgggat1141 ttcctgcaga aagacttgaa ggcgtataca ggaacaatat tgatgatgta gtaaggtttt1201 tggattcaaa gcataaaaac cattacaaga tatacaatct ttgtgctgaa agacattatg1261 acaccgccaa atttaattgc agagttgcac aatatccttt tgaagaccat aacccaccac1321 agctagaact tatcaaaccc ttttgtgaag atcttgacca atggctaagt gaagatgaca1381 atcatgttgc agcaattcac tgtaaagctg gaaagggacg aactggtgta atgatatgtg1441 catatttatt acatcggggc aaatttttaa aggcacaaga ggccctagat ttctatgggg1501 aagtaaggac cagagacaaa aagggagtaa ctattcccag tcagaggcgc tatgtgtatt1561 attatagcta cctgttaaag aatcatctgg attatagacc agtggcactg ttgtttcaca1621 agatgatgtt tgaaactatt ccaatgttca gtggcggaac ttgcaatcct cagtttgtgg1681 tctgccagct aaaggtgaag atatattcct ccaattcagg acccacacga cgggaagaca1741 agttcatgta ctttgagttc cctcagccgt tacctgtgtg tggtgatatc aaagtagagt1801 tcttccacaa acagaacaag atgctaaaaa aggacaaaat gtttcacttt tgggtaaata1861 cattcttcat accaggacca gaggaaacct cagaaaaagt agaaaatgga agtctatgtg1921 atcaagaaat cgatagcatt tgcagtatag agcgtgcaga taatgacaag gaatatctag1981 tacttacttt aacaaaaaat gatcttgaca aagcaaataa agacaaagcc aaccgatact2041 tttctccaaa ttttaaggtg aagctgtact tcacaaaaac agtagaggag ccgtcaaatc2101 cagaggctag cagttcaact tctgtaacac cagatgttag tgacaatgaa cctgatcatt2161 atagatattc tgacaccact gactctgatc cagagaatga accttttgat gaagatcagc2221 atacacaaat tacaaaagtc tgaatttttt tttatcaaga gggataaaac accatgaaaa2281 taaacttgaa taaactgaaa atggaccttt ttttttttaa tggcaatagg acattgtgtc2341 agattaccag ttataggaac aattctcttt tcctgaccaa tcttgtttta ccctatacat2401 ccacagggtt ttgacacttg ttgtccagtt gaaaaaaggt tgtgtagctg tgtcatgtat2461 ataccttttt gtgtcaaaag gacatttaaa attcaattag gattaataaa gatggcactt2521 tcccgtttta ttccagtttt ataaaaagtg gagacagact gatgtgtata cgtaggaatt2581 ttttcctttt gtgttctgtc accaactgaa gtggctaaag agctttgtga tatactggtt2641 cacatcctac ccctttgcac ttgtggcaac agataagttt gcagttggct aagagaggtt2701 tccgaagggt tttgctacat tctaatgcat gtattcgggt taggggaatg gagggaatgc2761 tcagaaagga aataatttta tgctggactc tggaccatat accatctcca gctatttaca2821 cacacctttc tttagcatgc tacagttatt aatctggaca ttcgaggaat tggccgctgt2881 cactgcttgt tgtttgcgca ttttttttta aagcatattg gtgctagaaa aggcagctaa2941 aggaagtgaa tctgtattgg ggtacaggaa tgaaccttct gcaacatctt aagatccaca3001 aatgaaggga tataaaaata atgtcatagg taagaaacac agcaacaatg acttaaccat3061 ataaatgtgg aggctatcaa caaagaatgg gcttgaaaca ttataaaaat tgacaatgat3121 ttattaaata tgttttctca attgtaacga cttctccatc tcctgtgtaa tcaaggccag3181 tgctaaaatt cagatgctgt tagtacctac atcagtcaac aacttacact tattttacta3241 gttttcaatc ataatacctg ctgtggatgc ttcatgtgct gcctgcaagc ttcttttttc3301 tcattaaata taaaatattt tgtaatgctg cacagaaatt ttcaatttga gattctacag3361 taagcgtttt ttttctttga agatttatga tgcacttatt caatagctgt cagccgttcc3421 acccttttga ccttacacat tctattacaa tgaattttgc agttttgcac attttttaaa3481 tgtcattaac tgttagggaa ttttacttga atactgaata catataatgt ttatattaaa3541 aaggacattt gtgttaaaaa ggaaattaga gttgcagtaa actttcaatg ctgcacacaa3601 aaaaaagaca tttgattttt cagtagaaat tgtcctacat gtgctttatt gatttgctat3661 tgaaagaata gggttttttt tttttttttt tttttttttt ttaaatgtgc agtgttgaat3721 catttcttca tagtgctccc ccgagttggg actagggctt caatttcact tcttaaaaaa3781 aatcatcata tatttgatat gcccagactg catacgattt taagcggagt acaactacta3841 ttgtaaagct aatgtgaaga tattattaaa aaggtttttt tttccagaaa tttggtgtct3901 tcaaattata ccttcacctt gacatttgaa tatccagcca ttttgtttct taatggtata3961 aaattccatt ttcaataact tattggtgct gaaattgttc actagctgtg gtctgaccta4021 gttaatttac aaatacagat tgaataggac ctactagagc agcatttata gagtttgatg4081 gcaaatagat taggcagaac ttcatctaaa atattcttag taaataatgt tgacacgttt4141 tccatacctt gtcagtttca ttcaacaatt tttaaatttt taacaaagct cttaggattt4201 acacatttat atttaaacat tgatatatag agtattgatt gattgctcat aagttaaatt4261 ggtaaagtta gagacaacta ttctaacacc tcaccattga aatttatatg ccaccttgtc4321 tttcataaaa gctgaaaatt gttacctaaa atgaaaatca acttcatgtt ttgaagatag4381 ttataaatat tgttctttgt tacaatttcg ggcaccgcat attaaaacgt aactttattg4441 ttccaatatg taacatggag ggccaggtca taaataatga cattataatg ggcttttgca4501 ctgttattat ttttcctttg gaatgtgaag gtctgaatga gggttttgat tttgaatgtt4561 tcaatgtttt tgagaagcct tgcttacatt ttatggtgta gtcattggaa atggaaaaat4621 ggcattatat atattatata tataaatata tattatacat actctcctta ctttatttca4681 gttaccatcc ccatagaatt tgacaagaat tgctatgact gaaaggtttt cgagtcctaa4741 ttaaaacttt atttatggca gtattcataa ttagcctgaa atgcattctg taggtaatct4801 ctgagtttct ggaatatttt cttagacttt ttggatgtgc agcagcttac atgtctgaag4861 ttacttgaag gcatcacttt taagaaagct tacagttggg ccctgtacca tcccaagtcc4921 tttgtagctc ctcttgaaca tgtttgccat acttttaaaa gggtagttga ataaatagca4981 tcaccattct ttgctgtggc acaggttata aacttaagtg gagtttaccg gcagcatcaa5041 atgtttcagc tttaaaaaat aaaagtaggg tacaagttta atgtttagtt ctagaaattt5101 tgtgcaatat gttcataacg atggctgtgg ttgccacaaa gtgcctcgtt tacctttaaa5161 tactgttaat gtgtcatgca tgcagatgga aggggtggaa ctgtgcacta aagtgggggc5221 tttaactgta gtatttggca gagttgcctt ctacctgcca gttcaaaagt tcaacctgtt5281 ttcatataga atatatatac taaaaaattt cagtctgtta aacagcctta ctctgattca5341 gcctcttcag atactcttgt gctgtgcagc agtggctctg tgtgtaaatg ctatgcactg5401 aggatacaca aaaataccaa tatgatgtgt acaggataat gcctcatccc aatcagatgt5461 ccatttgtta ttgtgtttgt taacaaccct ttatctctta gtgttataaa ctccacttaa5521 aactgattaa agtctcattc ttgtcaaaaa aaaaaaaaaa aaaaaaaaaa aa

Mutations in the PTEN protein can also be detected as is known in theart. The protein sequence of PTEN is found at GenBank Accession No.NP_(—)000305.3 GI:73765544, hereby incorporated by reference, and shownhere as SEQ ID NO:6:

  1 mtaiikeivs rnkrryqedg fdldltyiyp niiamgfpae rlegvyrnni ddvvrfldsk 61 hknhykiynl caerhydtak fncrvaqypf edhnppqlel ikpfcedldq wlseddnhva121 aihckagkgr tgvmicayll hrgkflkaqe aldfygevrt rdkkgvtips qrryvyyysy181 llknhldyrp vallfhkmmf etipmfsggt cnpqfvvcql kvkiyssnsg ptrredkfmy241 fefpqplpvc gdikveffhk qnkmlkkdkm fhfwvntffi pgpeetsekv engslcdqei301 dsicsierad ndkeylvltl tkndldkank dkanryfspn fkvklyftkt veepsnpeas361 sstsvtpdvs dnepdhyrys dttdsdpene pfdedqhtqi tkv

In another embodiment, a method for identifying a HER2-positive cancerpatient suitable for treatment with a 4-anilinoquinazoline kinaseinhibitor and an AKT inhibitor, comprising: (a) measuring the expressionlevel of the SASH1 gene in a sample from the patient; and (b) comparingthe expression level of said gene from the patient with the expressionlevel of the gene in a normal tissue sample or a reference expressionlevel (such as the average expression level of the gene in a cell linepanel or a cancer cell or tumor panel, or the like), wherein an increasein the expression level of SASH1 indicates the patient is suitable fortreatment with the 4-anilinoquinazoline kinase inhibitor and an AKTinhibitor.

In another embodiment, a method for identifying a HER2-positive cancerpatient suitable for treatment with a 4-anilinoquinazoline kinaseinhibitor and an AKT inhibitor, comprising: (a) obtaining the sequenceof the PIK3CA gene from a sample from a patient; (b) identifying anymutations in the PIK3CA gene in a sample from the patient as compared tothe wild-type sequence found in SEQ ID NO: 3, 4, or 11, (c) measuringthe expression level of the SASH1 gene in the sample from the patient;and (d) comparing the expression level of said gene from the patientwith the expression level of the gene in a normal tissue sample or areference expression level (such as the average expression level of thegene in a cell line panel or a cancer cell or tumor panel, or the like),wherein both a mutation in the PIK3CA gene and an increase in theexpression level of SASH1 indicates the patient is suitable fortreatment with the 4-anilinoquinazoline kinase inhibitor.

The expression level of the SASH1 gene is measured by measuring theamount or number of molecules of mRNA or transcript in a cell. Themeasuring can comprise directly measuring the mRNA or transcriptobtained from a cell, or measuring the cDNA obtained from an mRNApreparation thereof. Such methods of extracting the mRNA or transcriptfrom a cell, or preparing the cDNA thereof are well known to thoseskilled in the art. In other embodiments, the expression level of a genecan be measured by measuring or detecting the amount of protein orpolypeptide expressed, such as measuring the amount of antibody thatspecifically binds to the protein in a dot blot or Western blot. Theproteins described in the present invention can be overexpressed andpurified or isolated to homogeneity and antibodies raised thatspecifically bind to each protein. Such methods are well known to thoseskilled in the art.

The SASH1 protein (also known as KIAA0790, O94885, PEPE1, Q8TAI0,Q9H7R7, and SASH 1) is a poorly described protein so named because ofSAM and SH3 domains contained in the protein [GenBank Accession NumberNM_(—)015278.3 GI:45935384, GenBank mRNA accession number CCDS5212.1,and protein sequence O94885.3 GI:145559526]. SASH1 has been implicatedas a putative tumor suppressor gene in both breast and lung cancer. Itis thought to be involved in signal transduction, and recent evidencesuggests it may be involved in PI3K pathway signaling. The expressionlevel of a gene encoding SASH1 can be measured using an oligonucleotidederived from the nucleotide sequence of GenBank Accession No.NM_(—)015278.3 GI:45935384, hereby incorporated by reference, and shownhere as SEQ ID NO: 7:

   1 attttgaaga gaggggtccc ggggagctcc ctccaagatc tagaggctcc gcggccaccc  61 ctgccgggtc ctgccaagac ttgctagaag gaacgagtcg cgtgccttag ttagttggtt 121 cccgtcacag gaagaaacgc ctttgcagtg ggtttaattg cttctgggcc gagcgaattc 181 cccgccgtac aactcagtgg tgcggacttt gcctcctgct accctgttgc tgcgccgagc 241 ggggtgggaa agtttctgga gttgtcagtc gcgcagcccg tggccaccta gacccgaggt 301 gcgggcgcct gcgaagggcc cccgcggggt ggccggggcc gccggggcat gcagcgcggg 361 ggcgcggctc ggtgacgccg cgggcgggga cccggcatcc gggcaggctg cgcgcgggtg 421 cggggcgagg gcgccgcggg gactgggacg cacggcccgc gcgcgggaca cggccatgga 481 ggacgcggga gcagctggcc cggggccgga gcctgagccc gagcccgagc cggagcccga 541 gcccgcgccg gagccggaac cggagcccaa gccgggtgct ggcacatccg aggcgttctc 601 ccgactctgg accgacgtga tgggtatcct ggacggttca ctgggaaaca tcgatgacct 661 ggcgcagcag tatgcagatt attacaacac ctgtttctcc gacgtgtgcg agaggatgga 721 ggagctgcgg aaacggcggg tttcccagga cctggaagtg gagaaacccg atgctagccc 781 cacgtcactt cagctgcggt cccagatcga agagtcgctt ggcttctgta gcgccgtgtc 841 aaccccagaa gtggaaagaa agaaccctct tcataaatca aactcagaag acagctctgt 901 aggaaaagga gactggaaga agaaaaataa gtatttctgg cagaacttcc gaaagaacca 961 gaaaggaata atgagacaga cttcaaaagg agaagacgtt ggttatgttg ccagtgaaat1021 aacgatgagc gatgaggagc ggattcagct aatgatgatg gtcaaagaaa agatgatcac1081 aattgaggaa gcacttgcta ggctcaagga atacgaggcc cagcaccggc agtcggctgc1141 cctggaccct gctgactggc cagatggttc ttacccaacg tttgatggct catcaaactg1201 caattcaaga gaacaatcgg atgatgagac tgaggagtcg gtgaagttta agaggttaca1261 caagctggta aactccactc gcagagtcag aaagaaacta attagggtgg aagaaatgaa1321 aaaacccagc actgaaggtg gggaggagca cgtgtttgag aattcgccgg tcctggatga1381 acggtccgcc ctctactctg gcgtgcacaa gaagcccctt ttctttgatg gctctcctga1441 gaaacctccc gaagatgact cagactctct caccacgtct ccatcctcca gcagcctgga1501 cacctggggg gctggccgga agttggtcaa aaccttcagc aaaggagaga gccggggcct1561 gattaagccc cccaagaaga tggggacatt cttctcctac ccagaagaag aaaaggccca1621 gaaagtgtcc cgctccctca ccgaggggga gatgaagaag ggtctcgggt ccctaagcca1681 cgggagaacc tgcagttttg gaggatttga cttgacgaat cgctctctgc acgttggcag1741 taataattct gacccaatgg gtaaagaagg agactttgtg tacaaagaag tcatcaaatc1801 acctactgcc tctcgcatct ctcttgggaa aaaggtgaaa tcagtgaaag agacgatgag1861 aaagagaatg tctaaaaaat acagcagctc tgtctctgag caggactcgg gccttgatgg1921 aatgcctggc tcccctccgc cttcacagcc cgaccccgaa cacttggaca agcccaagct1981 caaggccggg ggttctgtag aaagtcttcg cagttctctc agtgggcaga gctccatgag2041 cggtcaaaca gtgagcacca ctgattcctc aaccagcaac cgggaaagcg tcaagtcgga2101 agatggggat gacgaagagc cgccttaccg aggcccgttc tgcgggcgtg ccagggtgca2161 caccgacttc acccccagtc cctatgacac agactcactc aagctcaaga aaggagatat2221 catcgatata atcagcaagc cacccatggg gacctggatg ggcctgctga acaacaaagt2281 cggcacgttc aagttcatct acgtggacgt gctcagtgaa gacgaggaga aacccaaacg2341 ccccaccagg aggcgtcgga aaggacgacc accccagccc aagtctgtgg aggatctcct2401 ggatcggatt aacctaaaag agcacatgcc cactttcctg ttcaatggat atgaagattt2461 ggacaccttt aagctgctgg aggaggaaga cttggatgag ttaaatatca gggacccgga2521 acacagagct gttctcttga cagcagtgga gctgttacaa gagtatgaca gtaacagcga2581 ccagtcagga tcccaggaga agctgctcgt tgacagccag ggcctgagtg gatgctcacc2641 ccgagactca ggatgctacg aaagcagtga gaacctggaa aacggcaaga ctcggaaagc2701 tagcctccta tctgccaagt catccaccga gcccagcttg aagtctttta gcagaaacca2761 gttgggcaat tacccaacat tgcctttaat gaaatcaggg gatgcactga agcagggaca2821 ggaggagggc aggctgggtg gtggccttgc cccagacacg tccaagagct gtgacccacc2881 tggtgtgact ggtttgaata aaaaccgaag aagcctccca gtttccatct gccggagctg2941 tgagaccctg gagggccccc agactgtgga cacttggccc cgatcccatt ccctggatga3001 ccttcaagtg gagcctggtg ctgagcaaga cgtgcctacc gaggtgacag aaccgccccc3061 tcagattgta cctgaagtgc cacagaagac gaccgcctct tccacgaagg cccagcccct3121 ggagcaagac tctgctgtcg acaatgcatt gctactgacc caaagcaaga gattttctga3181 acctcagaaa ttgacaacta agaaactgga gggctcaatc gcagcctctg gtcgcggcct3241 gtcaccccct cagtgtttgc ccagaaacta tgatgctcag cctcctggag ctaaacacgg3301 tttagcaagg acgcctctgg agggccacag aaaaggacac gagtttgaag gaacacacca3361 tcccctgggc accaaagaag gggtagatgc tgagcagaga atgcagccca aaattccatc3421 acagcctcca cctgttcctg ccaaaaagag cagagaacgc cttgctaacg gactccaccc3481 tgttcccatg ggccccagtg gggccctccc cagtcccgat gcgccatgcc tgccagtgaa3541 aaggggcagc cccgccagcc ccaccagccc tagcgactgt cccccagcac tggctcccag3601 gcctctctca gggcaggcgc ctggcagccc accaagcaca aggccgcccc cctggctctc3661 agagctcccc gagaacacaa gcctccagga gcacggtgtg aagctgggcc cggctttgac3721 caggaaggtc tcctgtgccc ggggagtgga tctagaaacg ctcactgaaa acaagctgca3781 cgctgaaggc atcgatctca cggaggagcc gtattctgat aagcatggcc gctgtgggat3841 tcctgaagcc ctggtgcaga gatacgcaga ggacttggat cagcccgagc gggacgtcgc3901 cgccaacatg gaccagatcc gggtgaagca gcttcggaag cagcaccgca tggcgattcc3961 aagtggtgga ctcacggaaa tctgccgaaa gcccgtctct cctgggtgca tttcgtctgt4021 gtcagattgg ctcatttcca tcggtctgcc catgtacgcc ggcaccctct ccaccgcggg4081 cttcagcaca ctgagccaag tgccttctct gtctcacact tgccttcagg aggccggcat4141 cacagaggag agacacataa gaaagctcct atctgcagcc agactcttca aactgccgcc4201 aggccctgag gccatgtagc caggcccgga atgggcctct ctggacaaga gccacccttt4261 cactgtgcat atgatgctga tgcaattcct ccatcatctc tggacgtgca gaccagatcc4321 agaagaaagg cctggcgtgt ggccaaacag cgtgaaacct tggcacagga ctgaggatcc4381 tctcctccag aaaagccccc tcgaggaaat aaattagtgc ggttctcttt gacccccaaa4441 gacaagacaa gcacttattt ttattttcag aagacaaaag aaccaagatg ccaactggct4501 gcgaatgctc tatctccagt ctgtctctgt gtactggtag aggctgggag gagtaggggg4561 cagcctgttc catttctgat agtgcccttg ctcttctgtc tgtcatcttg caggatgccc4621 gagggccaga tgggcttagc taggccaaag taacagactc aagagttatt gtacattact4681 gaccacgctc atttgttcaa aagttagaac atctggctgc accaggaaaa aaaaaaaaaa4741 aaagtcctgt tcttctttag ataaacaaga gacattttca taattgcttt ctagcaatca4801 gcttttattt gccttaatat aagcttttaa gcagttatct aactagtgtc cacaaccctg4861 taaccatact tccacatctt cagcttaggc agacatcgaa cctctctggg atgtttccag4921 caaaagtgag cttttctaat cgtctcattg taacatggct tattttgtag aggtattcat4981 cagccacaca cttcatgttg gtttttggtt tttaagctaa ctacaaatct agtaaaaagc5041 tatctgaaat tcacaaatat catgtgtgtg cgtgcgtgcg tgcgcgtgtg tgtctgtatt5101 catagtgact gcttttggtt ttaaccagtt tagtatcgtt actgtgtgga tcgtcgcgct5161 gcagtattga cttggaatcc tgaccatgtc catcccaaaa ttcagtcctc agttaacgga5221 tcatgtttgc aaaaggtcac tgtgaggctg catatttcag aaagatgtcc ttaataaggg5281 aagtcatgta taagatgttt tctaaaagac ttttcagtat tacaactaat actattatta5341 tccttctttt tttatttaga taattctttt aatttaaaca aaggttcact atggaaccag5401 acaaatctca ttagccatgt gttaagtatt tgctacttta aattgtttta caactgattt5461 cagcacattc tatccttttt tttttttgaa atggagtttc gctcttgtca cccaggctgg5521 agtgcaatgg cacgatcttg gctcactgca acctcagtct cccaggttca agtgattctc5581 ctgccttagc ctcccgagta gctgggatta taggcaccca ccaccacgcc cagctaattt5641 ttgtattatt agtagagaca gggtttcacc atgttggcca ggctggtctc aaactcaact5701 cctgacctca ggtggtccac ccgcctcagc ctcccaaagt gctgggatta caggtgtgag5761 ccaccgcacc tggcctctgt cctcttttag tctagtgtct ggttttctag caaacagtaa5821 atttaaacaa gtaaactatt atggtttcca ttgcttacaa aatgattttc ctttacattc5881 ttatcatgaa cactatttta agcatcaaat gcaatcatct aaaatataaa ggtcaatcat5941 ttataataga aacaccttga ccacaagccc ttgattgaac attttataat atttcatcta6001 cttattaaaa caaataattt cccttgggtt ggaggggaag tgatttcata aattaattag6061 aaagccatct ttagcatatt gcttatgtct ggatccatgt ttctgaggaa aaagacattc6121 tcaggtgatg tatttttttc atgcattagt atgcattttt aaaaaataat gcatgtttct6181 ttaataatta attttcatct tctataagat gccatgtgaa gaagttgtgg aaatgtagaa6241 taaaaagcta aagctgccaa atttctgttg aactcttaaa aacagctcat gtttgtttgt6301 cctctcgggt tgtggcctag cctatttgca atgtaatgaa gctgcagggt tcttgtatag6361 ctaaagcgtt caatgcattt cacgtgctgt ggtggatgtg ggtgctgtag acaggcttct6421 tctcttcctg ctctcaaaat acctcggctt gacatttgga cagatcctgt cattgtttaa6481 gctgagcaaa aaaccacaca aaagttgtgt aagagatgag ataacaaagg agcgagagaa6541 atctcatgtg aatttccaag ttttaattcg ttctccatga aggattttca tttcagtgaa6601 agtcgcagca gaagagggaa ctttctggag tttttgagaa tgccaaacca catttttatc6661 acacttcttt ggaaatcaat gcctttgcat agaaaatcaa attcagggac cacaaagaat6721 tttcagtggg aatgtctagt ctgaggggtc tgaggttgtt tttactttat tgtgttgttt6781 aaatatttta aaaatatctt tagcgtttgg tctttttttt ttctgtaaac atttaatttg6841 gtctgagaaa agctgaatgt ttgggtgtga cgtttgactg aggtggattg gggctgcctg6901 tggacattag tgaacaggtg gtaggcttca ggaatatcca gttttaatca gttgcatttg6961 gtacagaatt ttgagtaatg gtgaaaattg ttgtctttgg aaagcacaaa agaaacctgg7021 aaaggcagtt cggctcaggt agctacacat aacattgtgt atgattttca cttcaaagct7081 gtctggaagg aaatgcagtc agctccagct agtactattt atgtacccag ataactaaga7141 tattgtttca tggccttgcc ttagtcagag gcccttttct ctgtcctgaa cccccaggta7201 tgggtgaaat tggaaattac taatctattg gaaatcagtt cctgacatag taaagtttgc7261 tttcataact gcagcaaaaa aggtcaactt gccaagtcac tgctgccatg tgtgtactgt7321 attattttca gaaaaaaata taatagtctg agtccaagtt atcttgattt aaaattgata7381 gagaaaagaa actgtcgagc aagttatata acaactaaca acattgcact ttctgtatat7441 gaaatcaata tttaaataac ttatttttct ccattgctgt tcttaaaaac attgtaagta7501 gctgtaatat accagtacca atatgttctt gcaattgctt cagcccaaga aagctgtgta7561 ttgttttaaa aattgtaaaa attattgtga tgattcattt agcataaaga gaggtggacg7621 gaagggtttt cctatgtatc aaaacttgtc tataattatg tcatctatgt acctagaaaa7681 aagtaaataa atttcttcag ttgaatatg

In some embodiments, SASH1 protein levels can be detected and comparedto normal or reference levels. The SASH1 protein sequence is found atprotein sequence GenBank Accession No. O94885.3 GI:145559526, herebyincorporated by reference, and shown here as SEQ ID NO:8:

   1 medagaagpg pepepepepe pepapepepe pkpgagtsea fsrlwtdvmg ildgslgnid  61 dlaqqyadyy ntcfsdvcer meelrkrrvs qdlevekpda sptslqlrsq ieeslgfcsa 121 vstpeverkn plhksnseds svgkgdwkkk nkyfwqnfrk nqkgimrqts kgedvgyvas 181 eitmsdeeri qlmmmvkekm itieealarl keyeaqhrqs aaldpadwpd gsyptfdgss 241 ncnsreqsdd eteesvkfkr lhklvnstrr vrkklirvee mkkpstegge ehvfenspvl 301 dersalysgv hkkplffdgs pekppeddsd slttspssss ldtwgagrkl vktfskgesr 361 glikppkkmg tffsypeeek aqkvsrslte gemkkglgsl shgrtcsfgg fdltnrslhv 421 gsnnsdpmgk egdfvykevi ksptasrisl gkkvksvket mrkrmskkys ssyseqdsgl 481 dgmpgsppps qpdpehldkp klkaggsves lrsslsgqss msgqtvsttd sstsnresvk 541 sedgddeepp yrgpfcgrar vhtdftpspy dtdslklkkg diidiiskpp mgtwmgllnn 601 kvgtfkfiyv dvlsedeekp krptrrrrkg rppqpksved lldrinlkeh mptflfngye 661 dldtfkllee edldelnird pehravllta vellqeydsn sdqsgsgekl lvdsqglsgc 721 sprdsgcyes senlengktr kasllsakss tepslksfsr nqlgnyptlp lmksgdalkq 781 gqeegrlggg lapdtskscd ppgvtglnkn rrslpvsicr scetlegpqt vdtwprshsl 841 ddlqvepgae qdvptevtep ppqivpevpq kttasstkaq pleqdsavdn allltqskrf 901 sepqklttkk legsiaasgr glsppqclpr nydaqppgak hglartpleg hrkghefegt 961 hhplgtkegv daeqrmqpki psqpppvpak ksrerlangl hpvpmgpsga lpspdapclp1021 vkrgspaspt spsdcppala prplsgqapg sppstrpppw lselpentsl qehgvklgpa1081 ltrkvscarg vdletltenk lhaegidlte epysdkhgrc gipealvqry aedldqperd1141 vaanmdqirv kqlrkqhrma ipsgglteic rkpvspgcis svsdwlisig lpmyagtlst1201 agfstlsqvp slshtclqea giteerhirk llsaarlfkl ppgpeam

In some embodiments of the invention, the method further comprisesadministering a therapeutically effective amount of the4-anilinoquinazoline kinase inhibitor to the patient. Compounds andformulations of 4-anilinoquinazoline kinase inhibitors suitable for usein the present invention, and the dosages and methods of administrationthereof, are taught in U.S. Pat. Nos. 6,391,874; 6,713,485; 6,727,256;6,828,320; and 7,157,466, and International Patent Application Nos.PCT/EP97/03672, PCT/EP99/00048, and PCT/US01/20706 (which areincorporated in their entireties by reference).

In some embodiments of the invention, the lapatinib is lapatinibditosylate monohydrate. Lapatinib ditosylate monohydrate is commerciallyavailable under the brand name TYKERB® (GlaxoSmithKline; ResearchTriangle Park, NC). The prescription information of TYKERB® (FullPrescribing Information, revised March 2007, GlaxoSmithKline), which isincorporated in its entirety by reference, teaches one method ofadministration of the lapatinib to the patient.

The AKT gene is Homo sapiens v-akt murine thymoma viral oncogene homolog1 (AKT1) on chromosome 14. The genomic sequence for AKT is found atGenBank Accession No. NG 012188.1 GI:237874257, hereby incorporated byreference and identified as SEQ ID NO:12. The serine-threonine proteinkinase encoded by the AKT1 gene is catalytically inactive inserum-starved primary and immortalized fibroblasts. AKT1 and the relatedAKT2 are activated by platelet-derived growth factor. The activation israpid and specific, and it is abrogated by mutations in the pleckstrinhomology domain of AKT 1. It was shown that the activation occursthrough phosphatidylinositol 3-kinase. In the developing nervous systemAKT is a critical mediator of growth factor-induced neuronal survival.Survival factors can suppress apoptosis in a transcription-independentmanner by activating the serine/threonine kinase AKT1, which thenphosphorylates and inactivates components of the apoptotic machinery.Multiple alternatively spliced transcript variants have been found forthis gene and are collectively referred to here as AKT. Homo sapiensv-akt murine thymoma viral oncogene homolog 1 (AKT1), transcript variant2, mRNA at GenBank Accession No: NM_(—)001014432.1 GI:62241014, herebyincorporated by reference, is shown here as SEQ ID NO:9:

   1 cggcaggacc gagcgcggca ggcggctggc ccagcgcagc cagcgcggcc cgaaggacgg  61 gagcaggcgg ccgagcaccg agcgctgggc accgggcacc gagcggcggc ggcacgcgag 121 gcccggcccc gagcagcgcc cccgcccgcc gcggcctcca gcccggcccc gcccagcgcc 181 ggcccgcggg gatgcggagc ggcgggcgcc ggaggccgcg gcccggctag gcccgcgctc 241 gcgcccggac gcggcggccc ggggcttagg gaaggccgag ccagcctggg tcaaagaagt 301 caaaggggct gcctggagga ggcagcctgt cagctggtgc atcagaggct gtggccaggc 361 cagctgggct cggggagcgc cagcctgaga ggagcgcgtg agcgtcgcgg gagcctcggg 421 caccatgagc gacgtggcta ttgtgaagga gggttggctg cacaaacgag gggagtacat 481 caagacctgg cggccacgct acttcctcct caagaatgat ggcaccttca ttggctacaa 541 ggagcggccg caggatgtgg accaacgtga ggctcccctc aacaacttct ctgtggcgca 601 gtgccagctg atgaagacgg agcggccccg gcccaacacc ttcatcatcc gctgcctgca 661 gtggaccact gtcatcgaac gcaccttcca tgtggagact cctgaggagc gggaggagtg 721 gacaaccgcc atccagactg tggctgacgg cctcaagaag caggaggagg aggagatgga 781 cttccggtcg ggctcaccca gtgacaactc aggggctgaa gagatggagg tgtccctggc 841 caagcccaag caccgcgtga ccatgaacga gtttgagtac ctgaagctgc tgggcaaggg 901 cactttcggc aaggtgatcc tggtgaagga gaaggccaca ggccgctact acgccatgaa 961 gatcctcaag aaggaagtca tcgtggccaa ggacgaggtg gcccacacac tcaccgagaa1021 ccgcgtcctg cagaactcca ggcacccctt cctcacagcc ctgaagtact ctttccagac1081 ccacgaccgc ctctgctttg tcatggagta cgccaacggg ggcgagctgt tcttccacct1141 gtcccgggag cgtgtgttct ccgaggaccg ggcccgcttc tatggcgctg agattgtgtc1201 agccctggac tacctgcact cggagaagaa cgtggtgtac cgggacctca agctggagaa1261 cctcatgctg gacaaggacg ggcacattaa gatcacagac ttcgggctgt gcaaggaggg1321 gatcaaggac ggtgccacca tgaagacctt ttgcggcaca cctgagtacc tggcccccga1381 ggtgctggag gacaatgact acggccgtgc agtggactgg tgggggctgg gcgtggtcat1441 gtacgagatg atgtgcggtc gcctgccctt ctacaaccag gaccatgaga agctttttga1501 gctcatcctc atggaggaga tccgcttccc gcgcacgctt ggtcccgagg ccaagtcctt1561 gctttcaggg ctgctcaaga aggaccccaa gcagaggctt ggcgggggct ccgaggacgc1621 caaggagatc atgcagcatc gcttctttgc cggtatcgtg tggcagcacg tgtacgagaa1681 gaagctcagc ccacccttca agccccaggt cacgtcggag actgacacca ggtattttga1741 tgaggagttc acggcccaga tgatcaccat cacaccacct gaccaagatg acagcatgga1801 gtgtgtggac agcgagcgca ggccccactt cccccagttc tcctactcgg ccagcggcac1861 ggcctgaggc ggcggtggac tgcgctggac gatagcttgg agggatggag aggcggcctc1921 gtgccatgat ctgtatttaa tggtttttat ttctcgggtg catttgagag aagccacgct1981 gtcctctcga gcccagatgg aaagacgttt ttgtgctgtg ggcagcaccc tcccccgcag2041 cggggtaggg aagaaaacta tcctgcgggt tttaatttat ttcatccagt ttgttctccg2101 ggtgtggcct cagccctcag aacaatccga ttcacgtagg gaaatgttaa ggacttctgc2161 agctatgcgc aatgtggcat tggggggccg ggcaggtcct gcccatgtgt cccctcactc2221 tgtcagccag ccgccctggg ctgtctgtca ccagctatct gtcatctctc tggggccctg2281 ggcctcagtt caacctggtg gcaccagatg caacctcact atggtatgct ggccagcacc2341 ctctcctggg ggtggcaggc acacagcagc cccccagcac taaggccgtg tctctgagga2401 cgtcatcgga ggctgggccc ctgggatggg accagggatg ggggatgggc cagggtttac2461 ccagtgggac agaggagcaa ggtttaaatt tgttattgtg tattatgttg ttcaaatgca2521 ttttgggggt ttttaatctt tgtgacagga aagccctccc ccttcccctt ctgtgtcaca2581 gttcttggtg actgtcccac cgggagcctc cccctcagat gatctctcca cggtagcact2641 tgaccttttc gacgcttaac ctttccgctg tcgccccagg ccctccctga ctccctgtgg2701 gggtggccat ccctgggccc ctccacgcct cctggccaga cgctgccgct gccgctgcac2761 cacggcgttt ttttacaaca ttcaacttta gtatttttac tattataata taatatggaa2821 ccttccctcc aaattcttca ataaaagttg cttttcaaaa aaaaaaaaaa aaaaaaaa

The AKT protein found as GenBank Accession No. NP_(—)001014432.1GI:62241015, and hereby incorporated by reference is shown here at SEQID NO:10:

  1 msdvaivkeg wlhkrgeyik twrpryfllk ndgtfigyke rpqdvdqrea plnnfsvaqc 61 qlmkterprp ntfiirclqw ttviertfhv etpeereewt taiqtvadgl kkqeeeemdf121 rsgspsdnsg aeemevslak pkhrvtmnef eylkllgkgt fgkvilvkek atgryyamki181 lkkevivakd evahtltenr vlqnsrhpfl talkysfqth drlcfvmeya nggelffhls241 rervfsedra rfygaeivsa ldylhseknv vyrdlklenl mldkdghiki tdfglckegi301 kdgatmktfc gtpeylapev ledndygrav dwwglgvvmy emmcgrlpfy nqdheklfel361 ilmeeirfpr tlgpeaksll sgllkkdpkq rlgggsedak eimqhrffag ivwqhvyekk421 lsppfkpqvt setdtryfde eftaqmitit ppdqddsmec vdserrphfp qfsysasgta

It is contemplated that the AKT inhibitor can target AKT in various waysincluding targeting the AKT gene (SEQ ID NO:12), AKT mRNA (SEQ ID NO: 9)expression or expression levels, or the AKT protein (SEQ ID NO:10) orprotein levels,

Small Molecule Inhibitors.

In some embodiments of the invention, the AKT inhibitor is a smallmolecule kinase inhibitor, more specifically a serine/threonine kinaseinhibitor. In one embodiment, compounds and formulations of an AKTkinase inhibitor include small molecule protein kinase inhibitors suchas an aminofuruzan. An aminofuruzan suitable for use in the presentinvention, and the dosages and methods of administration thereof,include GSK690693 and other compounds taught in U.S. Pat. Nos.7,157,476; 7,348,339; and 7,547,779, and International PatentApplication No. PCT/US2004/027182, hereby incorporated by reference. Inother embodiments, the AKT kinase inhibitor suitable for use in thepresent invention, and the dosages and methods of administrationthereof, include heterocyclic carboxamide compounds such as GSK2141795and other compounds taught in WO2008/098104 and WO/2010/093885, both ofwhich are hereby incorporated by reference. These heterocycliccarboxamide compounds are inhibitors of the activity of one or more ofthe isoforms of the serine/threonine kinase, Akt. Some of the compoundsof described are inhibitors of the activity of all three isoforms of theserine/threonine kinase, Akt.

In one example, we used a novel AKT inhibitor developed byGlaxoSmithKline (GSK690693), which is an ATP-competitive, low-nanomolarpan-Akt kinase inhibitor. It is selective for the three Akt isoforms,although it does inhibit other members of the AGC kinase family.

In another example, we used a novel AKT inhibitor developed byGlaxoSmithKline (GSK2141795), which is a heterocyclic carboxamidecompound. that is an inhibitor of the activity of one or more of theisoforms of the serine/threonine kinase, Akt.

In other embodiments, the AKT inhibitor can be a therapeutic includingbut not limited to, antisense or inhibitory oligonucleotides, RNAinterference, siRNAs, aptamers, monoclonal antibodies, and smallmolecules.

Antibodies.

In one embodiment, a method of treatment using a humanized monoclonalAKT antibody to down-regulate AKT. Specific antibodies can be made bygeneral methods known in the art. A preferred method of generating theseantibodies is by first synthesizing peptide fragments. These peptidefragments should likely cover the coding gene region. Since synthesizedpeptides are not always immunogenic by their own, the peptides should beconjugated to a carrier protein before use. Appropriate carrier proteinsinclude but are not limited to Keyhole limpet hemacyanin (KLH). Theconjugated peptides should then be mixed with adjuvant and injected intoa mammal, preferably a rabbit through intradermal injection, to elicitan immunogenic response. Samples of serum can be collected and tested byELISA assay to determine the titer of the antibodies and then harvested.

Polyclonal (e.g., anti-AKT) antibodies can be purified by passing theharvested antibodies through an affinity column. Monoclonal antibodiesare preferred over polyclonal antibodies and can be generated accordingto standard methods known in the art of creating an immortal cell linewhich expresses the antibody.

Nonhuman antibodies are highly immunogenic in human and that limitstheir therapeutic potential. In order to reduce their immunogenicity,nonhuman antibodies need to be humanized for therapeutic application.Through the years, many researchers have developed different strategiesto humanize the nonhuman antibodies. One such example is using“HuMAb-Mouse” technology available from MEDAREX, Inc. and disclosed byvan de Winkel, in U.S. Pat. No. 6,111,166 and hereby incorporated byreference in its entirety. “HuMAb-Mouse” is a strain of transgenic micewhich harbor the entire human immunoglobin (Ig) loci and thus can beused to produce fully human monoclonal antibodies.

Inhibitor Oligonucleotides and RNA Interference (RNAi).

The approaches to be taken will depend on the detailed characteristicsof the genes, but in some embodiments, will begin with strategies toinhibit RNA transcription since they can, in principal, be used toattack over expressed genes independent of their biochemicalcomposition. Work in the past two decades on transcriptional inhibitorsfocused on oligodeoxynucleotides and ribozymes. These approaches havehad some clinical success but delivery issues limited their clinicalutility. Recently, however, advances in short interfering RNA (siRNA)technology and biological understanding have accelerated development ofanti-gene therapies (Wall, N. R. & Shi, Y. Small RNA: can RNAinterference be exploited for therapy? Lancet 362, 1401-3 (2003);Scanlon, K. J. Anti-genes: siRNA, ribozymes and antisense. Curr PharmBiotechnol 5, 415-20 (2004); Buckingham, S. D., Esmaeili, B., Wood, M. &Sattelle, D. B. RNA interference: from model organisms towards therapyfor neural and neuromuscular disorders. Hum Mol Genet. 13 Spec No 2,R275-88 (2004)). Promising therapeutic approaches include siRNAscomplexed with cationic liposomes (Liao, Y., et al., Enhanced paclitaxelcytotoxicity and prolonged animal survival rate by a nonviral-mediatedsystemic delivery of E1A gene in orthotopic xenograft human breastcancer. Cancer Gene Ther 11, 594-602 (2004); Yano, J. et al. Antitumoractivity of small interfering RNA/cationic liposome complex in mousemodels of cancer. Clin Cancer Res 10, 7721-6 (2004)), virusvector-mediated RNAi (Zhao, N. et al. Knockdown of Mouse Adultbeta-Globin Gene Expression in MEL Cells by Retrovirus Vector-MediatedRNA Interference. Mol Biotechnol 28, 195-200 (2004); Sumimoto, H. et al.Gene therapy for human small-cell lung carcinoma by inactivation ofSkp-2 with virally mediated RNA interference. Gene Ther (2004)) andnanoparticles adapted for siRNA (Schiffelers, R. M. et al. Cancer siRNAtherapy by tumor selective delivery with ligand-targeted stericallystabilized nanoparticle. Nucleic Acids Res 32, e149 (2004)). In oneembodiment, siRNAs against the high priority targets complexed withcationic liposomes and small molecule approaches to inhibit the overexpressed candidate genes will allow rapid development of this line ofattack.

In some embodiments, the expression of AKT is manipulated. In oneembodiment, such manipulation can be made using optimized siRNAs. SeeHannon, G. J. RNA interference (2002); Plasterk, R. H. in Science 1263-5(2002); and Elbashir, S. M. et al. in Nature 494-8 (2001). StrongPearson correlations between target gene amplification/expression levelsand pro-apoptotic effects of siRNAs will indicate that copynumber/expression levels determine the extent of apoptotic responses totarget gene inhibitors. Spearman rank test correlations betweenamplification detected and the level of induced apoptosis will indicateresponse to the targeted therapeutics.

High throughput methods can be used to identify AKT inhibitors such assiRNA and/or small molecular inhibitor formulations to deliver AKT (andother) inhibitors efficiently to cultured cells and xenografts. AKT (andother) inhibitory formulations will be preferentially effective againstxenografts that are amplified at the target loci and that these willenhance response to 4-anilinoquinazoline kinase inhibitor compounds.Effective formulations using such methods as described herein can bedeveloped for clinical application.

In one embodiment, RNA interference is used to generate smalldouble-stranded RNA (small interference RNA or siRNA) inhibitors toaffect the expression of a candidate gene generally through cleaving anddestroying its cognate RNA. Small interference RNA (siRNA) is typically19-22 nt double-stranded RNA. siRNA can be obtained by chemicalsynthesis or by DNA-vector based RNAi technology. Using DNA vector basedsiRNA technology, a small DNA insert (about 70 bp) encoding a shorthairpin RNA targeting the gene of interest is cloned into a commerciallyavailable vector. The insert-containing vector can be transfected intothe cell, and expressing the short hairpin RNA. The hairpin RNA israpidly processed by the cellular machinery into 19-22 nt doublestranded RNA (siRNA). In a preferred embodiment, the siRNA is insertedinto a suitable RNAi vector because siRNA made synthetically tends to beless stable and not as effective in transfection.

siRNA can be made using methods and algorithms such as those describedby Wang L, Mu F Y. (2004) A Web-based Design Center for Vector-basedsiRNA and siRNA cassette. Bioinformatics. (In press); Khvorova A,Reynolds A, Jayasena S D. (2003) Functional siRNAs and miRNAs exhibitstrand bias. Cell. 115(2):209-16; Harborth J, Elbashir S M, VandenburghK, Manning a H, Scaringe S A, Weber K, Tuschl T. (2003) Sequence,chemical, and structural variation of small interfering RNAs and shorthairpin RNAs and the effect on mammalian gene silencing. AntisenseNucleic Acid Drug Dev. 13(2):83-105; Reynolds A, Leake D, Boese Q,Scaringe S, Marshall W S, Khvorova A. (2004) Rational siRNA design forRNA interference. Nat. Biotechnol. 22(3):326-30 and Ui-Tei K, Naito Y,Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K.(2004) Guidelines for the selection of highly effective siRNA sequencesfor mammalian and chick RNA interference. Nucleic Acids Res.32(3):936-48, which are hereby incorporated by reference.

Other tools for constructing siRNA sequences are web tools such as thesiRNA Target Finder and Construct Builder available from GenScript(http://www.genscript.com), Oligo Design and Analysis Tools fromIntegrated DNA Technologies (URL:<http://www.idtdna.com/SciTools/SciTools.aspx>), or siDESIGN™ Centerfrom Dharmacon, Inc. (URL:<http://design.dharmacon.com/default.aspx?source=0>). siRNA aresuggested to built using the ORF (open reading frame) as the targetselecting region, preferably 50-100 nt downstream of the start codon.Because siRNAs function at the mRNA level, not at the protein level, todesign an siRNA, the precise target mRNA nucleotide sequence may berequired. Due to the degenerate nature of the genetic code and codonbias, it is difficult to accurately predict the correct nucleotidesequence from the peptide sequence. Additionally, since the function ofsiRNAs is to cleave mRNA sequences, it is important to use the mRNAnucleotide sequence and not the genomic sequence for siRNA design,although as noted in the Examples, the genomic sequence can besuccessfully used for siRNA design. However, designs using genomicinformation might inadvertently target introns and as a result the siRNAwould not be functional for silencing the corresponding mRNA.

Rational siRNA design should also minimize off-target effects whichoften arise from partial complementarity of the sense or antisensestrands to an unintended target. These effects are known to have aconcentration dependence and one way to minimize off-target effects isoften by reducing siRNA concentrations. Another way to minimize suchoff-target effects is to screen the siRNA for target specificity.

In one embodiment, the siRNA can be modified on the 5′-end of the sensestrand to present compounds such as fluorescent dyes, chemical groups,or polar groups. Modification at the 5′-end of the antisense strand hasbeen shown to interfere with siRNA silencing activity and therefore thisposition is not recommended for modification. Modifications at the otherthree termini have been shown to have minimal to no effect on silencingactivity.

It is recommended that primers be designed to bracket one of the siRNAcleavage sites as this will help eliminate possible bias in the data(i.e., one of the primers should be upstream of the cleavage site, theother should be downstream of the cleavage site). Bias may be introducedinto the experiment if the PCR amplifies either 5′ or 3′ of a cleavagesite, in part because it is difficult to anticipate how long the cleavedmRNA product may persist prior to being degraded. If the amplifiedregion contains the cleavage site, then no amplification can occur ifthe siRNA has performed its function.

In some embodiments, siRNA is designed based upon the mRNA sequence ofAKT, SEQ ID NO: 9.

In another embodiment, antisense oligonucleotides (“oligos”) can bedesigned to inhibit AKT and other candidate gene function. Antisenseoligonucleotides are short single-stranded nucleic acids, which functionby selectively hybridizing to their target mRNA, thereby blockingtranslation. Translation is inhibited by either RNase H nucleaseactivity at the DNA:RNA duplex, or by inhibiting ribosome progression,thereby inhibiting protein synthesis. This results in discontinuedsynthesis and subsequent loss of function of the protein for which thetarget mRNA encodes.

In some embodiments, antisense oligos are phosphorothioated uponsynthesis and purification, and are usually 18-22 bases in length. It iscontemplated that the AKT gene antisense oligos may have othermodifications such as 2′-O-Methyl RNA, methylphosphonates, chimericoligos, modified bases and many others modifications, includingfluorescent oligos.

In some embodiments, active antisense oligos should be compared againstcontrol oligos that have the same general chemistry, base composition,and length as the antisense oligo. These can include inverse sequences,scrambled sequences, and sense sequences. The inverse and scrambled arerecommended because they have the same base composition, thus samemolecular weight and Tm as the active antisense oligonucleotides.Rational antisense oligo design should consider, for example, that theantisense oligos do not anneal to an unintended mRNA or do not containmotifs known to invoke immunostimulatory responses such as fourcontiguous G residues, palindromes of 6 or more bases and CG motifs.

Antisense oligonucleotides can be used in vitro in most cell types withgood results. However, some cell types require the use of transfectionreagents to effect efficient transport into cellular interiors. It isrecommended that optimization experiments be performed by usingdiffering final oligonucleotide concentrations in the 1-5 μm range within most cases the addition of transfection reagents. The window ofopportunity, i.e., that concentration where you will obtain areproducible antisense effect, may be quite narrow, where above thatrange you may experience confusing non-specific, non-antisense effects,and below that range you may not see any results at all. In a preferredembodiment, down regulation of the targeted mRNA (e.g. AKT mRNA SEQ IDNO: 9) will be demonstrated by use of techniques such as northern blot,real-time PCR, cDNA/oligo array or western blot. The same endpoints canbe made for in vivo experiments, while also assessing behavioralendpoints.

For cell culture, antisense oligonucleotides should be re-suspended insterile nuclease-free water (the use of DEPC-treated water is notrecommended). Antisense oligonucleotides can be purified, lyophilized,and ready for use upon re-suspension. Upon suspension, antisenseoligonucleotide stock solutions may be frozen at −20° C. and stable forseveral weeks.

Aptamers.

In another embodiment, aptamer sequences which bind to specific RNA orDNA sequences can be made. Aptamer sequences can be isolated throughmethods such as those disclosed in U.S. Pat. Nos. 5,756,291; 5,843,653;and 7,329,742, which are hereby incorporated by reference.

It is contemplated that the sequences described herein may be varied toresult in substantially homologous sequences which retain the samefunction as the original. As used herein, a polynucleotide or fragmentthereof is “substantially homologous” (or “substantially similar”) toanother if, when optimally aligned (with appropriate nucleotideinsertions or deletions) with the other polynucleotide (or itscomplementary strand), using an alignment program such as BLASTN(Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.(1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410),and there is nucleotide sequence identity in at least about 80%,preferably at least about 90%, and more preferably at least about 95-98%of the nucleotide bases.

Methods of Treatment.

The AKT therapeutics of the present invention, such as the smallmolecule AKT inhibitor, also can be used to treat or prevent a varietyof disorders associated with reduced survival rate, especially asrelated to cancers. The AKT therapeutics are administered to a patientin an amount sufficient to elicit a therapeutic response in the patient(e.g., reduction of tumor size and growth rate, prolonged survival rate,reduction in concurrent cancer therapeutics administered to patient). Anamount adequate to accomplish this is defined as “therapeuticallyeffective dose or amount.”

The AKT inhibitors of the invention can be administered directly to amammalian subject using any route known in the art, including e.g., byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular, or intradermal), inhalation, transdermal application,rectal administration, or oral administration.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

Administration of the AKT inhibitors of the invention can be in anyconvenient manner, e.g., by injection, intratumoral injection,intravenous and arterial stents (including eluting stents), cather, oraladministration, inhalation, transdermal application, or rectaladministration. In some cases, the peptides and nucleic acids areformulated with a pharmaceutically acceptable carrier prior toadministration. Pharmaceutically acceptable carriers are determined inpart by the particular composition being administered (e.g., nucleicacid or polypeptide), as well as by the particular method used toadminister the composition. Accordingly, there are a wide variety ofsuitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed.,1989).

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector (e.g. peptide or nucleic acid)employed and the condition of the patient, as well as the body weight orsurface area of the patient to be treated. The size of the dose alsowill be determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular peptideor nucleic acid in a particular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of diseases or disorder associated with thedisease, the physician evaluates circulating plasma levels of thepolypeptide or nucleic acid, polypeptide or nucleic acid toxicities,progression of the disease (e.g., breast cancer), and the production ofantibodies that specifically bind to the peptide. Typically, the doseequivalent of a polypeptide is from about 0.1 to about 50 mg per kg,preferably from about 1 to about 25 mg per kg, most preferably fromabout 1 to about 20 mg per kg body weight. In general, the doseequivalent of a naked c acid is from about 1 μg to about 100 μg for atypical 70 kilogram patient, and doses of vectors which include a viralparticle are calculated to yield an equivalent amount of therapeuticnucleic acid.

For administration, the AKT inhibitor of the present invention can beadministered at a rate determined by the LD-50 of the polypeptide ornucleic acid, and the side-effects of the polypeptide or nucleic acid atvarious concentrations, as applied to the mass and overall health of thepatient. Administration can be accomplished via single or divided doses,e.g., doses administered on a regular basis (e.g., daily) for a periodof time (e.g., 2, 3, 4, 5, 6, days or 1-3 weeks or more).

In certain circumstances it will be desirable to deliver thepharmaceutical compositions comprising the AKT inhibitor therapiesdisclosed herein parenterally, intravenously, intramuscularly, or evenintraperitoneally as described in U.S. Pat. No. 5,543,158; U.S. Pat. No.5,641,515 and U.S. Pat. No. 5,399,363. Solutions of the active compoundsas free base or pharmacologically acceptable salts may be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468). In all cases the form must besterile and must be fluid to the extent that easy syringability exists.It must be stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

To date, most studies have been performed with siRNA formulated insterile saline or phosphate buffered saline (PBS) that has ioniccharacter similar to serum. There are minor differences in PBScompositions (with or without calcium, magnesium, etc.) andinvestigators should select a formulation best suited to the injectionroute and animal employed for the study. Lyophilized oligonucleotidesand standard or siSTABLE siRNAs are readily soluble in aqueous solutionand can be resuspended at concentrations as high as 2.0 mM. However,viscosity of the resultant solutions can sometimes affect the handlingof such concentrated solutions.

While lipid formulations have been used extensively for cell cultureexperiments, the attributes for optimal uptake in cell culture do notmatch those useful in animals. The principle issue is that the cationicnature of the lipids used in cell culture leads to aggregation when usedin animals and results in serum clearance and lung accumulation.Polyethylene glycol complexed-liposome formulations are currently underinvestigation for delivery of siRNA by several academic and industrialinvestigators, including Dharmacon, but typically require complexformulation knowledge. There are a few reports that cite limited successusing lipid-mediated delivery of plasmids or oligonucleotides inanimals.

Oligonucleotides can also be administered via bolus or continuousadministration using an ALZET mini-pump (DURECT Corporation). Cautionshould be observed with bolus administration as studies of antisenseoligonucleotides demonstrated certain dosing-related toxicitiesincluding hind limb paralysis and death when the molecules were given athigh doses and rates of bolus administration. Studies with antisense andribozymes have shown that the molecules distribute in a related mannerwhether the dosing is through intravenous (IV), subcutaneous (sub-Q), orintraperitoneal (IP) administration. For most published studies, dosinghas been conducted by IV bolus administration through the tail vein.Less is known about the other methods of delivery, although they may besuitable for various studies. Any method of administration will requireoptimization to ensure optimal delivery and animal health.

For bolus injection, dosing can occur once or twice per day. Theclearance of oligonucleotides appears to be biphasic and a fairly largeamount of the initial dose is cleared from the urine in the first pass.Dosing should be conducted for a fairly long term, with a one to twoweek course of administration being preferred. This is somewhatdependent on the model being examined, but several metabolic disorderstudies in rodents that have been conducted using antisenseoligonucleotides have required this course of dosing to demonstrateclear target knockdown and anticipated outcomes.

Liposomes.

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the administration of the AKT inhibitory peptides andnucleic acids of the present invention. In particular, the compositionsof the present invention may be formulated for delivery eitherencapsulated in a lipid particle, a liposome, a vesicle, a nanosphere,or a nanoparticle or the like.

The formation and use of liposomes is generally known to those of skillin the art (see for example, Couvreur et al., 1977; Couvreur, 1988;Lasic, 1998; which describes the use of liposomes and nanocapsules inthe targeted antibiotic therapy for intracellular bacterial infectionsand diseases). Recently, liposomes were developed with improved serumstability and circulation half-times (Gabizon & Papahadjopoulos, 1988;Allen and Choun, 1987; U.S. Pat. No. 5,741,516). Further, variousmethods of liposome and liposome like preparations as potential drugcarriers have been reviewed (Takakura, 1998; Chandran et al., 1997;Margalit, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S.Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No.5,795,587).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 m. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. For example, antibodies may beused to bind to the liposome surface and to direct the liposomes and itscontents to particular cell types. Carbohydrate determinants(glycoprotein or glycolipid cell-surface components that play a role incell-cell recognition, interaction and adhesion) may also be used asrecognition sites as they have potential in directing liposomes toparticular cell types.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998;Douglas et al., 1987). To avoid side effects due to intracellularpolymeric overloading, such ultrafine particles (sized around 0.1 m)should be designed using polymers able to be degraded in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet theserequirements are contemplated for use in the present invention. Suchparticles may be are easily made, as described (Couvreur et al., 1980;1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry etal., 1995 and U.S. Pat. No. 5,145,684).

Combination Therapy.

In some embodiments, the AKT inhibitor is administered in combinationwith a 4-anilinoquinazoline kinase inhibitor for treating a HER2+cancer, including breast cancer. For example, an inhibitory AKT siRNAmay be administered in conjunction with lapatinib.

The AKT inhibitor and the 4-anilinoquinazoline kinase inhibitor may beadministered simultaneously or sequentially. For example, the AKTinhibitor may be administered first, followed by lapatinib.Alternatively, the 4-anilinoquinazoline kinase inhibitor may beadministered first, followed by the AKT inhibitor. In some cases, theAKT inhibitor and 4-anilinoquinazoline kinase inhibitor are administeredin the same formulation. In other cases the AKT inhibitor and the4-anilinoquinazoline kinase inhibitor are administered in differentformulations. When the AKT inhibitor and the 4-anilinoquinazoline kinaseinhibitor are administered in different formulations, theiradministration may be simultaneous or sequential.

The average dose required to inhibit growth by 50% (GI50) for thelapatinib plus AKT combination as found in the Examples was 60 nM (range18 nM to 120 nM). In 4 out of 11 HER2 amplified cell lines, there wassignificant synergy as measured by combination index (upper 95% lessthan 1) at multiple drug concentrations, while one additional lineshowed significant synergy at a single concentration. Synergy wasobserved when cells were co-treated (i.e. simultaneously) with Lapatiniband the AKT inhibitor.

Kits.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain lyophilized primers to amplify andsequence PIK3CA. One or more additional containers may enclose elements,such as reagents or buffers, to be used in the assay. Such kits mayalso, or alternatively, contain a detection reagent as described abovethat contains a reporter group suitable for direct or indirect detectionof antibody binding.

Kits can also be supplied for therapeutic uses. Thus, the subjectcomposition of the present invention may be provided, usually in alyophilized form, in a container. For example, the 4-anilinoquinazolinekinase inhibitor and AKT inhibitors described herein are included in thekits with instructions for use, and optionally with buffers,stabilizers, biocides, or inert proteins. It may be desirable to includean inert extender or excipient to dilute the active ingredients, wherethe excipient may be present in from about 1 to 99% weight of the totalcomposition.

Example 1

Lapatinib is a dual inhibitor of EGFR/HER2. Recent evidence suggeststhat resistance to HER2 inhibition by lapatinib may be in part due tore-activation of PI3K-AKT signaling mediated by HER3. The purpose ofthis study was to screen lapatinib in combination with a pan-AKTinhibitor in a panel of HER2 amplified breast cancer cell lines todetermine if this dual inhibition had synergistic effects in preventingcell line growth.

We treated cell lines with lapatinib, AKTi, or a combination of the twoto determine if there were synergistic interactions between thesetargeted agents. Of 11 HER2 positive cell lines tested, four showedstrong evidence of synergy, while four cell lines showed little or nosynergy (and even some evidence of antagonism). The remaining three celllines showed an intermediate response. Of the four lines with synergy,all were mutant for PIK3CA, while all four non-synergistic lines werewild-type for PIK3CA. From microarray data, we identified two probe sets(representing one gene, SASH1) at a False Discover Rate of less than 1%that showed a significant association between expression and response.SASH1 has previously been implicated as a tumor suppressor gene inbreast, although at this point, it is unclear whether it plays anyfunctional role or is simply a marker for synergistic response tolapatinib and AKTi. Our work demonstrates that a combination oflapatinib plus and AKT inhibitor may be beneficial in HER2 positivepatients who also have PI3K pathway mutations. Care should be taken inscreening patients prior to treatment, as the combination was found tobe antagonistic in some cell lines that did not have PIK3CA mutations.SASH1 may be a useful screening tool in identifying such patients.

We used a panel of breast cancer cell lines that represent theheterogeneity seen in breast tumors for drug screening. Fixed numbers ofcells were plated in 96 well plates. Using a Biotek liquid handlingrobot, cells were treated with nine two-fold dilutions of an AKTinhibitor (GSK690693), a second drug (lapatinib), or a combination ofthe two. Response was measured using CTG assay as % Growth Inhibition.50% Growth Inhibition (GI50) was calculated. Synergistic interactionsbetween drugs was calculated by standard median-effect method using %inhibition (inhibitory concentration) readings and reported as acombination index using the Synergy module in R.

Of 11 HER2 positive cell lines tested, four showed strong evidence ofsynergy in more than half of the nine concentrations used. Five celllines showed little or no synergy (and even some evidence ofantagonism). The remaining two cell lines showed an intermediateresponse. Of the four lines with synergy, all were mutant for PIK3CA,while only one of the five lines that did not show strong synergy wasmutant for PIK3CA. Interestingly, this line did show significant synergyat one dose combination, suggesting that it was likely to be moresensitive to lapatinib+AKTi than the other four lines. From microarraydata, we identified two probe sets (representing one gene, SASH1) at aFalse Discover Rate of less than 5% that showed a significantassociation between expression and response. SASH1 has previously beenimplicated as a tumor suppressor gene in breast, although at this point,it is unclear whether it plays any functional role or is simply a markerfor synergistic response to lapatinib and AKTi.

Our work demonstrates that a combination of lapatinib plus and AKTinhibitor may be beneficial in HER2 positive patients who also have PI3Kpathway mutations. Care should be taken in screening patients prior totreatment, as the combination was found to be antagonistic in some celllines that did not have PIK3CA mutations. SASH1 may be a usefulscreening tool in identifying such patients.

Example 2

We have increased the number of HER2+ cell lines tested with thecombination of Lapatinib+AKT inhibitor (GSK690693) from 11 to 22. Thepattern that we observed in Example 1 above, i.e., synergy between theseagents was only seen in PI3K mutant lines, has been maintained (see FIG.4).

Example 3

We have tested 20/21 of the HER2 amplified cell lines (all but HCC202,which is currently being assessed) with a second AKT inhibitor(GSK2141795) in combination with lapatinib. We were able to calculatesynergy values for 19/20 of the cell lines (21-NT cell line failed inthe calculation). Cells were treated in the same manner with the samedosages of lapatinib and AKT inhibitor (GSK2141795) as described for thelapatinib plus AKT inhibitor GSK690693 above.

The results of this combination strongly mirror those observed with theAKT inhibitor, as 5/6 of the lines with significant synergy are mutantfor PI3K (see FIG. 5). In contrast, of the 13 cell lines that do notshow synergy, only 2 have PI3K mutations. Of these, one has a PTENmutation, which may have a different effect than PIK3CA mutations (themutation observed in synergistic lines), while the other line (BT474) isthe only sample ever described with a K111N mutation in PIK3CA, raisingquestions about the functional significance of the mutation.

Example 4

We have taken a non-synergistic cell line (EFM192A) that has wild-typePIK3CA and introduced an H1047R PIK3CA mutant by lentiviraltransduction.

The cell line EFM192A was transduced with a mutant H1047R PIK3CAlentiviral vector using standard techniques. Briefly, the lentiviralvector containing mutant PIK3CA was transfected into the cell line 293along with plasmids containing the viral gag and env genes forpackaging. Supernatant containing virus was collected at 48 hourspost-transfection and filtered to remove cell debris. Supernatant wasthen used to transducer EFM192A cells grown to 70% confluence. After 8hours of treatment of the cells with supernatant, the medium wasreplaced. Following one day of growth, a selective agent (blasticidin,10 ug/ml) was added to select for transduced cells. Cells were left inhigh levels of selection agent for two weeks (by this time, all of theuntranduced EFM192A cells treated with blasticidin were killed). Thecells were then maintained in lower levels of blasticidin (2-5 ug/ml) toensure continued presence of the lentiviral delivered gene. Twenty fourhours prior to treatment with lapatinib and AKT inhibitor (GSK690693),blasticidin was removed. The transduced cells were treated in the samemanner with the same doses of targeted therapeutic agents as describedabove

Referring now to FIG. 6, treatment of this altered line withLapatinb+AKTi (GSK690693) resulted in significant synergy, stronglysuggesting that PIK3CA mutations are not only markers of synergy, buthave a direct effect on response to the drug combination.

Example 5

Lapatinib is currently approved for use in patients with advanced HER2positive breast cancer in conjunction with capecitabine. Patientseligible for Lapatinib+AKT inhibitor therapy would be those HER2positive patients who also harbor either a PI3K pathway mutation (PIK3CAmutation and possibly PTEN mutation/deletion) or high levels ofexpression of SASH1. Paraffin embedded tumor blocks could be assessedfor mutations in PIK3CA or PTEN using standard sequencing approaches.SASH1 levels could be assessed by RT-PCR approaches from paraffinembedded tissue using primers spanning a short amplicon or byimmunohistochemical staining of tissue sections if suitable antibodiescan be identified. Patients who have PI3K pathway mutations and/or highlevels of SASH1 would then be candidates to receive combination therapyof Lapatinib plus the AKT inhibitor. Lapatinib is an orally bioavailablesmall molecule inhibitor, while an AKT inhibitor such as GSK690693 orGSK2141795 may be delivered orally or intravenously to the patient.

It is to be understood that the present invention is not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “agene” includes a plurality of such genes, and so forth.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

The present examples, methods, procedures, specific compounds andmolecules are meant to exemplify and illustrate the invention and shouldin no way be seen as limiting the scope of the invention. Any patents,publications, publicly available sequences mentioned in thisspecification are indicative of levels of those skilled in the art towhich the invention pertains and are hereby incorporated by reference tothe same extent as if each was specifically and individuallyincorporated by reference.

TABLE 1 PIK3CA PTEN Lap AKT L + A Cell Line mutation mutation GI50 GI50GI50 HCC202 p.E545K nd 0.068 0.015 0.018 AU565 wt wt 0.087 0.109 0.045MDAMB361 wt/p.E545K wt 0.106 0.063 0.035 UACC812 wt wt 0.106 0.041 0.043MDAMB175 wt wt 0.114 0.027 0.037 BT474 wt/p.K111N wt 0.116 0.089 0.050SKBR3 wt wt 0.163 0.054 0.046 SUM225 wt nd 0.245 0.293 0.083 HCC1569 wtp.K267fs*9 0.420 0.122 0.083 HCC1954 p.H1047R wt 0.673 0.119 0.085SUM190PT p.H1047R nd 0.806 0.229 0.119 MDAMB453 p.H1047R wt/p.E307K0.922 0.109 0.079

TABLE 2 List of cell lines used in the study (note: MDAMB175 is not HER2amplified and so was excluded from the synergy experiments) PIK3CA PTENCell Line Status ICBP43 Class status status p53 status 21MT-1 HER2amplified NO N/A wt N/A N/A 21MT-2 HER2 amplified NO N/A wt N/A N/A 21NTHER2 amplified NO N/A wt N/A N/A 21PT HER2 amplified NO N/A wt N/A N/AAU-565 HER2 amplified YES Luminal wt wt p.R175H BT-474 HER2 amplifiedYES Luminal wt/p.K111N wt p.E285K EFM192A HER2 amplified NO N/A wt N/AN/A EFM192B HER2 amplified NO N/A wt N/A N/A EFM192C HER2 amplified NON/A wt N/A N/A HCC1569 HER2 amplified YES BasalA wt p.K267fs * 9 p.E294*HCC1954 HER2 amplified YES BasalA p.H1047R wt wt/p.Y163 HCC202 HER2amplified YES Luminal p.E545K nd nd JIMT1 HER2 amplified NO N/A wt N/AN/A MDA-MB-175-VII HER2 over-expressing YES Luminal wt wt wt MDA-MB-361HER2 amplified YES Luminal wt/p.E545K wt wt MDA-MB-453 HER2 amplifiedYES Luminal p.H1047R wt/p.E307K wt SK-BR-3 HER2 amplified YES Luminal wtwt nd SUM190PT HER2 amplified NO N/A p.H1047R nd nd SUM225CWN HER2amplified NO Luminal wt nd nd UACC-812 HER2 amplified YES Luminal wt wtwt UACC-893 HER2 amplified YES Luminal p.H1047R wt p.R342* ZR-75-30 HER2amplified YES Luminal wt wt wt

We claim:
 1. A method for identifying a HER2-positive cancer patientsuitable for treatment with a 4-anilinoquinazoline kinase inhibitor andan AKT inhibitor, comprising: (a) obtaining the sequence of the PIK3CAgene, mRNA, or protein from a sample from a patient; and (b) identifyingany mutations in the PIK3CA gene in a sample from the patient ascompared to the wild-type sequence found in SEQ ID NOS:3, 4, or 11,wherein a mutation in the PIK3CA gene, mRNA or protein sequenceindicates the patient is suitable for treatment with the4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
 2. Themethod of claim 1, further comprising (c) measuring the expression levelof a gene encoding HER2 in a sample from the patient, and (d) comparingsaid expression level of the gene encoding HER2 in said patient to theexpression level of the gene encoding HER2 in a normal tissue sample, areference expression level, or the average expression level of HER2 in apanel of normal cell lines or cancer cell lines, wherein an increase inthe expression level of HER2 indicates the patient is suitable fortreatment with the 4-anilinoquinazoline kinase inhibitor.
 3. The methodof claim 1, wherein the mutation identified in the PIK3 CA gene or mRNAsequence from the patient sample in step (b) expresses a mutant PIK3CAprotein with a missense mutation found at amino acid residues 542-545and/or amino acid residues, 1047-1049.
 4. The method of claim 1, whereinthe mutation identified in the PIK3 CA protein from the patient samplein step (b) is a missense mutation found at amino acid residues 542-545and/or amino acid residues, 1047-1049.
 5. The method of claim 1, whereinthe cancer is breast cancer.
 6. The method of claim 1, furthercomprising administering a therapeutically effective amount of the4-anilinoquinazoline kinase inhibitor and an AKT inhibitor to thepatient.
 7. The method of claim 6, wherein the 4-anilinoquinazolinekinase inhibitor is a lapatinib.
 8. The method of claim 7, wherein thelapatinib is lapatinib ditosylate monohydrate.
 9. The method of claim 8,wherein the AKT inhibitor is an aminofuruzan.
 10. The method of claim 9,wherein the AKT inhibitor is GSK690693.
 11. The method of claim 6,wherein the AKT inhibitor is a heterocyclic carboxamide.
 12. The methodof claim 11, wherein the AKT inhibitor is GSK2141795.
 13. The method ofclaim 5, wherein the AKT inhibitor is an antisense or inhibitoryoligonucleotide, RNA interference, siRNA, a monoclonal antibody, or asmall molecule.
 14. The method of claim 2, wherein the HER2 expressionlevel can be detected by measuring the levels of SEQ ID NO: 2 in thepatient sample or by immunohistochemical analysis.
 15. A method foridentifying a HER2-positive cancer patient suitable for treatment with a4-anilinoquinazoline kinase inhibitor and an AKT inhibitor, comprising:(a) measuring the expression level of the SASH1 gene in a sample fromthe patient; and (b) comparing the expression level of said gene fromthe patient with the expression level of the gene in a normal tissuesample or a reference expression level (such as the average expressionlevel of the gene in a cell line panel or a cancer cell or tumor panel,or the like), wherein an increase in the expression level of SASH1indicates the patient is suitable for treatment with the4-anilinoquinazoline kinase inhibitor and the AKT inhibitor.
 16. Themethod of claim 15, wherein the cancer is breast cancer.
 17. The methodof claim 16, further comprising administering a therapeuticallyeffective amount of the 4-anilinoquinazoline kinase inhibitor and an AKTinhibitor to the patient.
 18. The method of claim 17, wherein the4-anilinoquinazoline kinase inhibitor is a lapatinib.
 19. The method ofclaim 18, wherein the lapatinib is lapatinib ditosylate monohydrate. 20.The method of claim 19, wherein the AKT inhibitor is an aminofuruzan.21. The method of claim 20, wherein the AKT inhibitor is GSK690693. 22.The method of claim 15, wherein the AKT inhibitor is a heterocycliccarboxamide.
 23. The method of claim 22, wherein the AKT inhibitor isGSK2141795.
 24. A method for identifying a HER2-positive cancer patientsuitable for treatment with a 4-anilinoquinazoline kinase inhibitor andan AKT inhibitor, comprising: (a) measuring the expression level of theSASH1 gene in a sample from the patient; and (b) comparing theexpression level of said gene from the patient with the expression levelof the gene in a normal tissue sample or a reference expression level,wherein an increase in the expression level of SASH1 indicates thepatient is suitable for treatment with the 4-anilinoquinazoline kinaseinhibitor and an AKT inhibitor.
 25. The method of claim 24, wherein theexpression level of the SASH1 gene is measured by detecting the levelsof SEQ ID NOs: 7 or
 8. 26. The method of claim 24, wherein the cancer isbreast cancer.
 27. The method of claim 24, further comprisingadministering a therapeutically effective amount of the4-anilinoquinazoline kinase inhibitor and an AKT inhibitor to thepatient.
 28. The method of claim 24, wherein the 4-anilinoquinazolinekinase inhibitor is a lapatinib.
 29. The method of claim 28, wherein thelapatinib is lapatinib ditosylate monohydrate.
 30. The method of claim24, wherein the AKT inhibitor is an antisense or inhibitoryoligonucleotide, RNA interference, siRNA, a monoclonal antibody, or asmall molecule.
 31. The method of claim 30, wherein the AKT inhibitor isa small molecule.
 32. The method of claim 31, wherein the AKT inhibitoris an aminofuruzan.
 33. The method of claim 32, wherein the AKTinhibitor is GSK690693.
 34. The method of claim 31, wherein the AKTinhibitor is a heterocyclic carboxamide.
 35. The method of claim 35,wherein the AKT inhibitor is GSK2141795.